Brother Paul was well known in the locality for his commitment to protecting the environment and the indigenous peoples.
"A law must be passed to eliminate forced conversions"
With no date set for a new election, Christians in Algeria know that the result of the elections is unpredictable.
In Burkina Faso, tensions periodically degenerate into violence between the Fulani and indigenous farmers.


Built from a used shipping crate as a guest house in San Antonio, this residence has sustainability in mind with every aspect of its design. Envisioned by Poteet Architects, the house sits on a platform made from old telephone poles, its used water is funneled to gardens on its roof, and it includes other nods to the environment like a composting toilet. With industrial origins and natural aspirations, the abode is full of exciting juxtapositions. To add to that theme, the interior of the house is fully finished in high style, with not a trace of the corrugated metal sides surrounding it. Bright colors are found throughout, including a captivating blue covering the exterior. While not forgetting its origins as a shipping crate, this home has an undoubtedly green nature.
The addition of wall-height windows brings in natural light to the interior of the shipping crate, while a patio deck off the front edge greatly extends its compact living space. On the top, a full rooftop garden is fed by waste water from the taps inside.
Despite its many residential additions and modifications, no attempt is made to hide the fact that the building is, in fact, a shipping container. The doors on one end and the retention of ID numbers along each side add to the charm of the cottage, marrying art and utility.
The entire container and its patio is raised a few inches above the lot, giving a step up into its spaces that helps separate it from the surrounding lawn. Color-matched plastic sheets are suspended over three metal supports to create an awning for the porch.
Half of the front window set at the living room doubles as a sliding door, the only entryway into the residence. Except for the bathroom window, each opening extends fully from floor to ceiling.
Inside, the main space of the guest house is a long, narrow living room. Due to the limitations of the eight-foot width of the container, the room's furniture is all arranged facing out the front windows.
Except for the width, it's almost impossible to tell from inside that this house originated as an industrial object. Wood paneling lines the walls and floors, and the decor is colorfully chic.
The dwelling's bathroom is totally lined in red, with a thin window at the top of one end. A final portion of the house beyond its bathroom is used as storage for gardening tools, opened on the left end of the structure from the two swinging container doors that were kept.


in the focused, personalized setting of a private lesson.
Contact Donna to learn more.
Donna Davis offers coaching sessions in a safe and comfortable place. Come away with techniques that will enhance professional effectiveness, strengthen confidence, and develop versatility of communication in all areas of life. Her proven resources and years of experience have helped people in a wide variety of contexts. Feel free to contact Donna with your questions.


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Rayane Hamidou (; born 13 April 2002) is a Saudi Arabian-Malawian professional footballer who plays as a right back for Al-Ahli.

Club career
Hamidou started his career at Al-Ahli and was called up to the first team in July 2021. On 16 August 2021, Hamidou signed his first professional contract with the club. He made his league debut for the club on 25 August 2021 by starting the match against Damac. On 29 January 2022, Hamidou joined Al-Tai on loan.

Honours

International
Saudi Arabia U23
WAFF U-23 Championship: 2022

References

External links

2002 births
Living people
Association football fullbacks
Saudi Arabian footballers
Malawian footballers
Sportspeople from Jeddah
Saudi Arabia youth international footballers
Saudi Professional League players
Saudi First Division League players
Al-Ahli Saudi FC players
Al-Tai FC players
Naturalised citizens of Saudi Arabia

redpajama_set_name: RedPajamaWikipedia


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Rachael Mattice, 24, was reported missing from a family campsite on Algonquin Lake on June 22.
According to reports, Mattice, of Wells, N.Y., was found alive five days later, about 50 miles from where she planned on going.
Rachel's friend Cynthia Nellis wrote a Facebook post regarding her disappearance.
According to Missing Persons of America, Mattice was found near her mother's home in Johnstown.
After her parent's didn't hear from Rachel, her father went to the camp and found her car but not his daughter.
Sheriff Karl Abrams told WRGB that Rachel is doing fine but at the moment can't say why she went missing in the first place.


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The Technical Centre for Agricultural and Rural Cooperation (CTA) shut down its activities in December 2020 at the end of its mandate. The administrative closure of the Centre was completed in November 2021.
ICT Update
Work with CTA
CTA Homepage
GODAN Action webinar: Agricultural Development - Open Data and the Sustainable Development Goals
The GODAN Action Webinar series address the need for capacity building among data users, producers and intermediaries to engage effectively with open data in the agriculture and nutrition sectors.
This webinar will focus on the use of open data to inform decision-making on sustainable development in Africa.
In Africa, the role of data, and more specifically open data, in informing national investment plans and associated multi-stakeholder consultations has been a clear part of the Comprehensive Africa Agricultural Development Program (CAADP) with the African Union working with partners to put in place a Regional Strategic Analysis and Knowledge Support System (ReSAKSS) on the continent for this purpose. As implementation of CAADP's current results framework enters its third year and the SDGs become mainstreamed in national agriculture strategies, it is now more important than ever to ensure stakeholders have access to the data and information that would allow them to participate in decision-making and hold each other to account.
Open data in agriculture and nutrition allows African governments to punch far above their weight by leveraging the wisdom of national experts in proposing adjustments to strategies and policies and also empowering stakeholders to design, implement and scale interventions that contribute to national targets. However, this requires the sharing of more data and information than that which is available from the national level SAKSS. It also requires, in some instances, changes to how data and information are managed, curated and shared within the public sector and beyond. How do we start strengthening open data initiatives to respond to this emerging reality and what would a roadmap look like?
Presenter: Muchiri Nyaggah, Executive Director– Local Development Research Institute
Muchiri has worked in technology and innovation consulting for twenty years, six of which focusing on open data and open government in Africa as a driver for improved development outcomes. He has keen interest in the implementation of Africa's development agenda, its impact and how developmental states can deliver better outcomes for Africa's people.
The Local Development Research Institute (LDRI) is an action-oriented think tank supporting efforts of African Union member states to end extreme poverty, end hunger and reduce inequalities. LDRI's work is focused on the role of agricultural transformation in these efforts, especially the capability of states to have available to them, and share openly, data and statistics for decision-making.
Wageningen, Netherlands
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Webinar: The future of food supply chains
Webinar: How blockchain can help smallholder farmers get access to digital idendity?
Webinar: The 9 ways IOT and blockchain can help smallholder farmers
Online course: Farm data management, sharing and services for agriculture development
GODAN Action webinar: Data ecosystem mapping to support FAIR data principles
Addison, Chris
Technical Centre for Agricultural and Rural Cooperation (CTA)
Msengezi, Chipo
GODAN supports the proactive sharing of open data to make information about agriculture and nutrition available, accessible and usable to deal with the urgent challenge of ensuring world food security. It is a rapidly growing group, currently with over 282 partners from national governments, non-governmental, international and private sector organisations that have committed to a joint Statement of Purpose. The initiative focuses on building high-level support among governments, policymakers, international organizations and business. GODAN promotes collaboration to harness the growing volume of data generated by new technologies to solve long-standing problems and to benefit farmers and the health of consumers. We encourage collaboration and cooperation between stakeholders in the sector.
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CTA is a joint institution operating under the framework of the Cotonou Agreement between the ACP Group of States (Africa, the Caribbean and the Pacific) and the EU Member States (European Union). CTA is funded by the European Union.
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redpajama_set_name: RedPajamaCommonCrawl


We propose a simple and scalable method for soft community detection that makes use of both graph structures and vertex attributes. Our method is based on micro-clustering, which is a scalable and efficient clique-based method for detecting overlapping communities in unweighted graphs. We extend this method to graphs with vertex attributes so that we can make use of information supplied by vertex attributes. Our method still requires the same time complexity as micro-clustering. We confirm the validity and efficiency of our method by applying it to a large-scale co-purchasing network of real online auction data.


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Archive for category: Uyghur Muslims
China, Forced Labor, Global Poverty, Human Rights, Minorities, Uyghur Muslims
Global Brands Connected to Forced Uyghur Labor
Forced labor stemming from human rights violations in the Xinjiang province of China has been linked to at least 83 major corporations. In a report released by the Australian Strategic Policy Institute (ASPI) in February 2020, companies such as Nike, Gap, H&M, Apple, BMW, Gap, Huawei, Samsung, Sony and Volkswagen all have connections to the use of forced Uyghur labor in China. The report identified 27 factories in China that employ the use of labor transferred from Xinjiang.
Human Rights Violations of the Uyghur Population
Between 2017 and 2019, it is estimated that over 80,000 Uyghurs were moved out of Xinjiang to work in factories across China through labor transfer programs known as "Xinjiang Aid." The Chinese government refers to these job assignments as "vocational training" while maintaining that they are part of the "re-education" process assigned to the Uyghur population. These programs have all been identified in connection to the human rights abuses of the Uyghur population as a whole.
It is reported that surveillance tools are being used to monitor the Uyghur population in these programs and to restrict their freedom of movement. Additionally, it has been reported that they are subject to threats, arbitrary detainment and abusive working conditions.
Factories Identified and Company Responses
The companies identified in connection to this forced labor use include international brands that span across the technology, clothing and automotive sectors.
In the technology sector, Apple, Amazon, Samsung, Sony and Microsoft, among others, have been connected to factories that utilize forced labor in China. Amazon has issued a statement saying they do not tolerate the use of forced labor and will be investigating these findings further.
The Qingdao Taekwang Shoes Co. Ltd has been specifically connected to forced labor of the Uyghur population. Workers at this factory also attend a night school that seems to closely resemble the "re-education camps" in the Xinjiang province. Nike is this factory's primary customer and released a statement saying that the factory has not recruited new workers from Xinjiang since last year and that it is seeking advice on the most responsible path toward handling the employment of the remaining workers from this region.
The Haoyuanpeng Clothing Manufacturing Co. Ltd is also identified as using forced labor. This factory's corporate website cites partnerships with the companies Fila, Adidas, Puma and Nike. Adidas specifically stated that it does not have a current relationship with the company and is investigating this claim. Nike has also released a statement that it has no current relationship with the factory.
Since the release of ASPI's report, H&M has ended a relationship with a Chinese yarn supplier due to its ties to forced labor.
The Global Supply Chain
The complexity of the global supply chain has undoubtedly made it more difficult for global corporations to monitor the connections of their suppliers to forced labor in China, but ASPI reached out to all 83 brands included in the report to confirm details of their suppliers as listed in the report.
Unfortunately, companies and consumers are now put at risk by purchasing goods that connect to forced labor. Investors in these 83 companies are potentially at risk as well. U.S. Congress has recently introduced legislation to protect investors through the requirement of disclosure of goods sourced from Xinjiang.
The End Uyghur Forced Labor Coalition
There are several advocacy groups dedicated to spreading awareness and furthering tangible steps to end the persecution and exploitation of the Uyghur population. The End Uyghur Forced Labor Coalition has written to 17 companies regarding the Uyghur Forced Labor Prevention Act (S. 3471), which is intended to end the use of forced labor from this region in supply chains. The coalition has also issued a call to action that aims for brands to remove all connections with suppliers that have used forced labor. This has been endorsed by investor organizations from more than 35 countries as well as more than 300 Uyghur groups, trade unions and civil society groups.
Ending Forced Uyghur Labor
Though most companies were not aware of the use of forced labor of Uyghurs, along with the awareness that was brought to light, action is also being taken by these companies to show that they do not support forced labor by any means. The End Uyghur Forced Labor Coalition is doing important work to continue bringing awareness to the issue and to protect the rights of this vulnerable minority population.
– Katherine Musgrave
https://borgenproject.org/wp-content/uploads/The_Borgen_Project_Logo_small.jpg 0 0 Lynsey Alexander https://borgenproject.org/wp-content/uploads/The_Borgen_Project_Logo_small.jpg Lynsey Alexander2021-02-16 07:30:442021-02-15 02:36:34Global Brands Connected to Forced Uyghur Labor
China, Global Poverty, Uighur, Uyghur Muslims
Being Uighur in Xinjiang
Beginning along the famed Silk Road's winding trails, the story of being Uighur in the Xinjiang territory in China is one of lost prosperity and an eternal struggle against the oppression from outside forces.
The Uighur Plight
At the height of the Karahanid Kingdom in 934 A.D., the Uighur were a prosperous people. Their cities were epicenters of philosophical and scientific thought, and the capital city Kashgar was a bastion of Islam. This all ended with the invasion of the Manchu Empire and the eventual takeover of the Chinese Nationalists in 1911.
Xinjiang has since been designated as an autonomous region within China. Despite this, the Chinese government has implemented numerous policies in hopes of assimilating the Uighur people and crushing separatist movements. The Uighurs have now become a minority in Xinjiang as the Han Chinese have become the majority in the region's urban areas. The Xinjiang have been abetted by government incentives, while the Uighurs have been largely confined to poor rural areas. The wealthy and influential capital city Urumqi is now approximately 75% Han Chinese.
Uighurs in Xinjiang have had their land redistributed to the Han migrants, leaving not enough farming land behind to make a living. According to Reuters, the Uighur people face discriminatory hiring practices with many businesses displaying signs banning them from applying for jobs. This marginalization along with growing poverty among the Uighur people has spurred increased resentment towards Beijing and the ruling Communist Party. In 2001, the Chinese government used 9/11 and the resulting American War on Terror to repress the Uighurs' desire for independence and begin intense surveillance and military operations.
Surveillance and Re-education
According to Human Rights Watch, Beijing requires officers in the region to use what is called the Integrated Joint Operations Platform (IJOP) as part of the Strike Hard Campaign to track the movements of the Uighurs and other ethnic minorities. The IJOP operating system and app, created by state-owned contractors, is used to aggregate data and flag the location of those deemed potentially threatening. The app tracks the movement of phones and vehicles, alerting officials to what are considered suspiciously long trips. The IJOP app also prompts officials to keep the biometric data of every person, including fingerprints, DNA, and blood type.
The IJOP has become a key component in the next stage in Beijing's ploy for control, particularly with the implementation of so-called "reeducation camps." Such camps were created by the Regulations on De-extremification in March 2017, specifically designed to convert Uighurs and other predominantly Muslim minorities to the ideological beliefs of the Communist Party. All forms of traditional religious clothing, literature, and practice are considered extremist and cause for internment in the camps under the regulation. Any form of travel is reason enough for being labeled suspicious and possibly being sent to the camps. People of all ages, male and female, are at risk. Security checks and invasive checks have become part of everyday life in Xinjiang, making it impossible to escape suspicion.
Inside the re-education camps, detainees are forced to learn about the teachings and ideologies of the Communist Party. According to those who have been detained, individuals who fail to comply are punished severely. The penalties range from verbal abuse to food deprivation, solitary confinement, beatings, and the use of restraints and stress positions. Deaths inside the camps have been reported but there is no way to verify how many people have died and the circumstances concerning their deaths. The number of detainees also remains unknown. Estimates are in the hundreds of thousands, possibly nearing one million.
What Is Being Done?
In the United Nations Committee on the Elimination of Racial Discrimination's (CERD) periodic report on China, the committee stated its concern that the poverty rates among ethnic minorities in Xinjiang remain high. CERD also stated its deep concern about "numerous reports of detention of large numbers of ethnic Uighurs and other Muslim minorities . . . without being charged or tried." CERD urged China to halt the unlawful detention of individuals and immediately release those who have been detained.
U.N. human rights chief Michelle Bachelet is currently seeking access to China to review these reports. However, Chinese officials claim the happiest Muslims in the world live in Xinjiang, as well as assert that "hostile Western forces" are simply misrepresenting and vilifying what is occurring in Xinjiang. The United States is preparing to enact a new round of sanctions against China over this mass imprisonment of Uighurs and other Muslim minorities. According to the Uyghur Humans Rights Project, these sanctions were previously halted due to trade negotiations with China during the G20 summit but have since been approved by all respective parties within the U.S. government.
Though the current situation for the Uighur people of Xinjiang remains dire, through diplomatic action by the U.N., the United States, and its allies are bringing awareness to the issue. Such dedication by international intervention has presented continued hope. Such hope is for a future where being Uighur in Xinjiang will cease to be a story of systematic oppression and instead will become a story of perseverance through great odds.
– Shane Thoma
https://borgenproject.org/wp-content/uploads/The_Borgen_Project_Logo_small.jpg 0 0 Lynsey Alexander https://borgenproject.org/wp-content/uploads/The_Borgen_Project_Logo_small.jpg Lynsey Alexander2021-02-14 12:22:212021-02-16 11:41:59Being Uighur in Xinjiang
China, Global Poverty, Islam, Muslims, Religion, Uyghur Muslims
Poverty & Oppression: The Uyghur Muslims in Xinjiang, China
More than 40 different ethnic groups live within China's Northwest region, known as Xinjiang. The two largest ethnic groups are the Han Chinese and Uyghur Muslims. The two groups do not speak the same language, nor do they share similar traditions. This creates a divide that widens due to the socio-economic disparity between the two factions. The Chinese government's treatment of the Uyghur Muslims in Xinjiang, through exploitation and other human rights violations, further exacerbated the issue. Unfortunately, fear of the Uyghurs has given the Chinese government a justification to detain and exploit millions.
The Issue of Poverty
The poverty in Xinjiang is most prevalent of any Chinese province at approximately 6%. However, certain regions suffer more than others. For example, Yutian County has a poverty rate of around 25%. Yet, the region has made great strides forward in poverty alleviation during recent years. In fact, more than 2.3 million people escaped poverty between 2014 and 2018. Xinjiang's resource-rich areas have caught the attention of the Han Chinese. This, in turn, drives migration and economic growth. Additionally, the government has promoted various industries, employment transfers and citizen relocation, further driving down poverty rates.
Despite this, many Uyghur Muslims in Xinjiang suffer exclusion from these benefits. Most prominently, employment discrimination prevents Uyghurs from obtaining jobs in these rising markets. As a result, a disproportionate amount of Han Chinese receives better jobs, furthering the economic disparity between the two groups. Furthermore, the rising number of Han Chinese in the region (currently at 40% of the population) has made the native Uyghurs feel distant from one another. The Uyghur Muslims in Xinjiang fear the loss of their culture.
Due to the exclusion and poverty that the Uyghur Muslims in Xinjiang experience, they tend to move closer to Islam. Some even go so far as to commit acts of violence. Regardless of the real reasons for the violence, many Han Chinese believe it is Islamic extremists causing violence. The Han Chinese believe this contributes to instability within the region as the Uyghur's are fighting for independence. This, in turn, leads to widespread fear and distrust among the population.
The Chinese government responded to these acts of violence by claiming that Islamic extremists caused it. Therefore, the government must "reeducate" the Uyghur Muslims in Xinjiang. Since 2014, China has suppressed the Uyghurs' culture, language and religion in the name of national security, while claiming that the Uyghurs have full freedom. Police stations now occupy every few blocks and cameras are on every street. Some public areas are inaccessible and many people are stopped on the street for identification. Notably, many Uyghurs have had their passports taken and can no longer leave the region.
Crackdown
Since 2017, the government has been detaining approximately 1 million Uyghurs in reeducation camps, with their only crimes being that they are Muslim. Hundreds of camps are present today with 39, having tripled in size from 2017 to 2018. Construction spending has increased drastically by nearly $3 billion in recent years.
Information on exact conditions in the camps is difficult to discern. However, previous detainees speak of a prison-like environment, sexual assault, forced abortions or contraceptives, extreme surveillance and torture. Some say they witnessed people taking their own lives.
Additionally, many Uyghurs in these camps must work in factories across China. They experience exploitation, completely against their will. The products they produce are widespread. Approximately 83 international companies use this forced labor in their supply chain. Moreover, 20% of cotton products around the world came from this forced labor.
Policy, Legislation and Coalition of Aid
Many U.S. companies benefit from this system. Legislation must pass to prevent forced labor and condemn China's actions. Most recently, the Senate passed the Uyghur Human Rights Policy Act of 2020 (S3744) in June 2020. It placed sanctions on many officials responsible or complicit in the detainment and abuse of the Uyghurs.
Particularly, the Uyghur Forced Labor Prevention Act emerged in March 2020 but has not passed into law yet. Additionally, many Uyghurs are stuck in U.S. immigration limbo, complicating their ability to seek refuge. Both proposals are crucial in helping significantly reduce the demand for forced labor. Further, people are urging the Chinese government to stop committing human rights abuses.
Many NGOs are working to bring attention to the crisis, as well as aiding the affected Uyghurs. Despite difficulties in offering direct aid, there exists a coalition of more than 250 organizations part of the End Uyghur Forced Labor campaign. The coalition demands companies eliminate any Uyghur forced labor within their production lines, within one year. Companies that agree must sign a pledge — applying pressure to all companies that have not yet signed. Also, the coalition organized advocacy days, began petitions and called on Congress to ban cotton from the Uyghur region. This additional pressure on companies will help end Uyghur forced labor. Hopefully, it will reduce demand for Uyghur labor and prevent their exploitation as well.
Hope Remains
Poverty in Xinjiang has reduced significantly. It will likely continue to decrease in the upcoming years. Additionally, numerous countries have applied pressure on the Chinese government. It is crucial that the U.S. does the same. Many NGOs have worked to raise awareness and apply pressure on governments and companies to eliminate Uyghur forced labor. Despite the many challenges that the Uyghurs have faced, hope remains for conditions to improve, with the support of the global community.
– Elizabeth Lee
https://borgenproject.org/wp-content/uploads/The_Borgen_Project_Logo_small.jpg 0 0 Kim Thelwell https://borgenproject.org/wp-content/uploads/The_Borgen_Project_Logo_small.jpg Kim Thelwell2020-11-02 07:30:202020-10-31 22:04:09Poverty & Oppression: The Uyghur Muslims in Xinjiang, China


There is ample evidence that crossing over is suppressed in heterochromatin associated with centromeres and nucleolus organizers (NORs). This characteristic has been attributed to all heterochromatin, but the generalization may not be justified. To investigate the relationship of crossing over to heterochromatin that is not associated with centromeres or NORs, we used a combination of fluorescence in situ hybridization of the maize 180-bp knob repeat to show the locations of knob heterochromatin and fluorescent immunolocalization of MLH1 protein and AFD1 protein to show the locations of MLH1 foci on maize synaptonemal complexes (SCs, pachytene chromosomes). MLH1 foci correspond to the location of recombination nodules (RNs) that mark sites of crossing over. We found that MLH1 foci occur at similar frequencies per unit length of SC in interstitial knobs and in the 1 µm segments of SC in euchromatin immediately to either side of interstitial knobs. These results indicate not only that crossing over occurs within knob heterochromatin, but also that crossing over is not suppressed in the context of SC length in maize knobs. However, because there is more DNA per unit length of SC in knobs compared to euchromatin, crossing over is suppressed (but not eliminated) in knobs in the context of DNA length compared to adjacent euchromatin.
BASED on observations of the liverwort Pellia epiphylla, Emil Heitz first named and described "heterochromatin" as chromatin that remains condensed throughout the cell cycle and "euchromatin" as chromatin that is decondensed during interphase (Heitz 1928). Subsequently, heterochromatin has been observed in a wide variety of other eukaryotes, and many additional characteristics have been attributed to heterochromatin, including suppression of meiotic crossing over (Green 1966; Yunis and Yasmineh 1971; Comings 1972; John 1976; Ris and Korenberg 1979; Stack 1984; Szauter 1984; Loidl 1987; Sumner 2003; Grewal and Jia 2007; Lichten 2008).
Constitutive heterochromatin (subsequently referred to simply as heterochromatin) is most commonly found at telomeres and nucleolus organizers (NORs) and to either side of centromeres (i.e., pericentric heterochromatin), but heterochromatin can occur anywhere on chromosomes (Hsu and Arrighi 1971; Yunis and Yasmineh 1971; Dumas and Britton-Davidian 2002; Neves et al. 2005). A variety of evidence indicates that meiotic crossing over is suppressed, if not completely eliminated, in heterochromatin at NORs and in pericentric heterochromatin. This is based on observations of chiasmata (Mather 1933; Brown 1949; Natarajan and Gropp 1971; John 1976; Dumas and Britton-Davidian 2002; Lukaszewski et al. 2012), comparisons of molecular linkage maps with sequenced pseudomolecules and chromosome structure (Petes and Botstein 1977; Kota et al. 1993; Castiglioni et al. 1999; Islam-Faridi et al. 2002; Yu et al. 2003; Kim et al. 2005; van Os et al. 2006; Gore et al. 2009; Tomato Genome Consortium 2012), and observations of recombination nodules (RNs) and MLH1 foci on synaptonemal complexes (SCs, pachytene chromosomes) (Carpenter 1975; Sherman and Stack 1995; Froenicke et al. 2002; Anderson et al. 2003; Marcon and Moens 2003).
A variety of hypotheses have been proposed to explain how crossing over is suppressed in heterochromatin (John and Lewis 1965; Yunis and Yasmineh 1971; Carpenter 1975; Miklos and Nankivell 1976; Stack 1984; Civardi et al. 1994; Schnable et al. 1998; Bennetzen 2000; Fu et al. 2002; Blitzblau et al. 2007; Lichten 2008; Ellermeier et al. 2010; Vader et al. 2011). One hypothesis is that DNA double-strand break formation and repair is inhibited in heterochromatin, possibly due to DNA and histone modifications (such as methylation and deacetylation), the association of certain protein factors and small interfering RNAs (siRNAs) with DNA, or steric exclusion of crossover enzymes and RNs. Other hypotheses focus on differences in chromosome cores and cohesins, SC structure in heterochromatin compared to euchromatin, underrepresentation of heterochromatin in SC length, and interference with synapsis (SC formation) in heterochromatin. Also, assuming that crossing over usually takes place in or near genes, differences in DNA sequence may contribute, including a high concentration of tandem repeats and/or a lack of genes in heterochromatin. Recently, Vincenten et al. (2015) provided strong support for two of these hypotheses by showing that, in Saccharomyces cerevisiae, a kinetochore (centromere) protein complex called Ctf19 both inhibits nearby double-strand breaks needed for crossing over and promotes cohesion enrichment that also interferes with meiotic crossing over.
Does demonstrated suppression of crossing over in pericentric and NOR heterochromatin justify the assumption that crossing over is suppressed in all heterochromatin regardless of its chromosomal position? While there is no definitive evidence one way or the other, the assumption is supported by a report in Drosophila virilis that little if any crossing over occurred in a block of pericentric heterochromatin that was translocated into distal euchromatin (Baker 1958). However, it is possible that centromere-related factors (such as Ctf19) remained associated with this translocated block of pericentric heterochromatin, and that this accounts for the continued suppression of crossing over. Indeed, a cause and effect relationship between condensed chromatin and suppression of crossing over is clouded by two types of observations. First, crossover suppression need not be mediated by heterochromatin. For example, centromeres without heterochromatin can suppress nearby crossing over (the "centromere effect"), as can NORs (Beadle 1932; Mather 1939; Petes and Botstein 1977; Yamamoto and Miklos 1978; Lambie and Roeder 1986; Resnick 1987; Kota et al. 1993; Choo 1998; Blitzblau et al. 2007; Lichten 2008; Vincenten et al. 2015). Furthermore, most of the maize genome is comprised of retrotransposons where little if any crossing over occurs, even though many of the retrotransposons are present in distal euchromatin (Fu et al. 2002; Yao et al. 2002). Also, in some Allium species that lack pericentric heterochromatin, crossing over is preferentially localized near centromeres and suppressed in distal euchromatin (Levan 1933; Albini and Jones 1988; Stack and Roelofs 1996). Second, heterochromatin tends to form at locations in chromosomes where crossing over is infrequent (Charlesworth et al. 1994), so it is possible that pericentric and NOR heterochromatin result from suppressed crossing over rather than cause it (Topp and Dawe 2006).
Crossing over in heterochromatin has been reported occasionally, most often from observations of chiasmata and RNs rather than by linkage mapping, because there are few genetic markers in heterochromatin and molecular markers are difficult to map in regions of low recombination (Loidl 1987; Latos-Bielenska et al. 1990; Sudman and Greenbaum 1990; Sherman and Stack 1995; Anderson et al. 2003). In most cases, chiasmata and RNs that appeared to be in heterochromatin were either illustrated without comment or interpreted as crossover events that actually occurred in euchromatin and only appeared to be in heterochromatin. In the latter case, one of the three following explanations was offered.
Chiasma terminalization. Chiasmata observed in distal heterochromatin were proposed to have formed in more proximal euchromatin and then moved distally, i.e., terminalized, until they stopped in distal heterochromatin (John 1976; Jones 1978). Chiasma terminalization was an idea popularized by Darlington (1937) to explain the apparent terminal location of chiasmata in many organisms. While chiasma terminalization was once widely accepted, it never had strong experimental support, and subsequent experiments in a variety of species using differential labeling of sister chromatids with tritiated thymidine, differential staining of sister chromatids with bromodeoxyuridine, and heterozygous subterminal C-bands provide convincing evidence that chiasmata do not terminalize (Jones 1987; Loidl 1987).
A problem with resolution. Chiasmata observed in heterochromatin were suggested to have formed in tiny islands of euchromatin within heterochromatin or in euchromatin near heterochromatin, so that subsequent chromatin condensation obscured the euchromatin (Linnert 1955; Fontana and Vickery 1974; Klášterská et al. 1974; Jones 1978; Loidl 1979; de la Torre et al. 1986; Berger and Greilhuber 1991). This is difficult to prove or disprove, and small islands of euchromatin have been observed in some heterochromatin (Linnert 1955; Ramanna and Prakken 1967). Based on the assumption that crossing over does not occur in heterochromatin, this is a plausible explanation for observations of chiasmata that appear to be in heterochromatin, but it is not evidence that crossing over does not occur in heterochromatin.
Heterochromatin stickiness. Bivalent associations in heterochromatin at late prophase I were proposed to be due not to chiasmata, but to "stickiness" of heterochromatin (Drets and Stoll 1974; Fontana and Vickery 1974; Godin and Stack 1976; John and King 1980, 1985; Cermeno 1984; Carlson 1988). Chromatin stickiness has been observed in both mitosis and meiosis, between both homologous and nonhomologous chromosomes, and between all combinations of euchromatin and heterochromatin (Rhoades 1955; Yunis and Yasmineh 1971; Godin and Stack 1975, 1976). Indeed, in Drosophila oocytes, stickiness between pairs of achiasmatic homologs substitutes for chiasmata to encourage proper segregation (Giauque and Bickel 2016). However, distinguishing between chromatin stickiness and chiasmata is often difficult because, except for a few groups of organisms (especially grasshoppers), chiasmata are not well-defined structurally (John 1976; Crolla and Polani 1989; Stack 1991). Consequently, in late prophase I, euchromatic connections between corresponding sites on homologs are generally accepted as chiasmata, while heterochromatic connections between corresponding sites on homologs may be dismissed as stickiness (Klášterská et al. 1974). Although this interpretation could be correct, it is a double standard based on the assumption that crossovers and chiasmata form only in euchromatin.
For these reasons, an investigation of the relationship between heterochromatin and crossover suppression should be carried out on homozygous dense masses of heterochromatin that lack interspersed islands of euchromatin, and which lie in euchromatin away from NORs and centromeres. Homozygous maize knobs meet these criteria because knobs are distinct masses of dense heterochromatin with no reported islands of euchromatin and knobs are located in distal, gene-rich euchromatin where crossing over is frequent (Buckler IV et al. 1999; Ghaffari et al. 2013). Knobs in maize are composed mainly of tandemly arranged blocks of a 180-bp repeat sequence interrupted by insertions of other repeated sequences including retrotransposons (Ananiev et al. 1998a).
In an earlier investigation of the distribution of RNs on maize SCs that were spread by hypotonic bursting, we found substantial numbers of RNs at the presumed sites of knobs and in euchromatin to either side of knobs (Figure 1) (Anderson et al. 2003). While we could not see knobs on spreads of maize SCs because the chromatin was dispersed, we knew where knobs were supposed to be based on the work of others (Chen et al. 2000). Because RNs occur at sites of crossing over (Carpenter 1975; Sherman and Stack 1995; Anderson et al. 2003, 2014; Marcon and Moens 2003), this result indicated that crossing over occurs along the SC in knob heterochromatin at rates similar to adjacent euchromatin. However, in deference to the assumption that crossing over is suppressed in heterochromatin, we suggested a model for the structure of homozygous knobs in which the knob on each homolog consists of a few long loops of condensed chromatin attached to a short segment of SC compared to the dimensions of the knob. The knob chromatin was proposed to mushroom over adjacent SC in euchromatin, so that most, if not all, RNs that appeared to be in knobs were really in euchromatin under an umbrella of knob heterochromatin (Figure 2).
Histograms showing the distribution of RNs along the length of KYS maize SCs 5, 6, 7, and 9. Each SC is represented on the x-axis in micrometers with the short arm to the left and the position of the kinetochore marked by a black dot. Beneath the x-axis, the locations of knobs are marked by horizontal, thick red lines, and the position of the NOR on SC 6 is marked by a horizontal, thin black line. The histogram bars on the y-axis represent the total number of RNs observed in each 0.2-μm segment of SC length for 203 SC 5's, 176 SC 6's, 178 SC 7's, and 234 SC 9's. A red smoothing line is superimposed over each histogram to show the general trend of RN distribution. These histograms are used with permission from GENETICS (Anderson et al. 2003). RN, recombination nodule; SC, synaptonemal complex.
Model to explain how crossing over could be suppressed in maize knob heterochromatin while RN frequencies on SC in knobs are similar to RN frequencies on SC in nearby euchromatin. In this frontal view of an SC, short, paired sister loops of euchromatin (orange) extend laterally from their regularly spaced attachment sites on lateral elements of the SC. Here, homozygous knob chromatin (black) consists of long, paired sister loops in each homolog that are collapsed to cover a length of SC well beyond their small attachment sites on the lateral elements. Thus, most of the SC covered by a knob is actually in euchromatin. A RN in the central region of the SC is shown located in euchromatin near the attachment sites of the knob loops. While this RN marks a crossover in euchromatin, it would look like the RN is in knob heterochromatin, thereby explaining our observations that RN frequencies "in knobs" are similar to RN frequencies in nearby euchromatin. This figure is used with permission from GENETICS (Anderson et al. 2003). RN, recombination nodule; SC, synaptonemal complex.
The research presented here tests our model by visualizing both knobs and crossover sites on the same spreads of SCs. This was accomplished by fluorescence in situ hybridization (FISH) of the 180-bp repeat found in maize knobs (Peacock et al. 1981; Ananiev et al. 1998a,b) and immunofluorescent labeling of maize SC lateral element protein AFD1 (Golubovskaya et al. 2006) and RN protein MLH1 to show crossover sites on SCs (Baker et al. 1996; Anderson et al. 1999, 2014). We found that long loops of knob heterochromatin attach to SCs along the entire length of knobs, not just at tiny sites, and that MLH1 foci occur on SCs within knob heterochromatin at frequencies similar to SCs in adjacent euchromatin.
KYS maize was grown in a greenhouse with controlled lighting and temperature.
Anthers 2.0–2.2 mm in length were fixed for 1–18 hr at room temperature in freshly prepared 1:3 acetic ethanol. Immediately after clearing in 45% acetic acid, anthers were transferred to a drop of 45% acetic acid on a glass microscope slide, where they were bisected with a microscalpel. Primary microsporocytes in pachytene were squeezed out with steel dissecting needles, anther walls were removed, and the cells were gently squashed under a siliconized cover glass. The cover glass was removed by the dry ice method, and squashes were air-dried for 1 hr. For bright field microscopy, a drop of 2% aceto-orcein was placed on the squashes and covered with a cover glass. The slide was gently heated (without boiling) over an alcohol flame and then allowed to cool. The slide was inverted over 95% ethanol until the cover glass fell off. Before the ethanol dried, a cover glass was mounted using a drop of Euparal. For fluorescence microscopy, a cover glass was mounted using 15 µl of Vectashield (Vector Laboratories, Burlingame, CA) containing 5 µg/ml 4′,6-diamidino-2-phenylindole (DAPI).
Details for SC spreading can be found in Stack and Anderson (2009). Briefly, 40–80 living anthers 2.0–2.2 mm in length were gently removed from florets to avoid damage, i.e., bruising and cytomixis, and placed in a depression slide containing 0.2 ml of a digestion medium that was 0.56 mM monobasic potassium phosphate, 0.8 mM CaCl2, 0.1 mM piperazine-N,N′-bis(ethane sulfonic acid) (acid PIPES), 0.2% potassium dextran sulfate (MW ∼10,000 Da), 1% polyvinylpyrrolidone, and 0.7 M mannitol, with the pH adjusted to 4.1 using 0.1 N HCl and/or 0.1 N KOH. The depression slide was incubated at 18–20° in a closed Y-Petri plate with 5–10 ml of water in the bottom. After 5 min, 3 mg of desalted, lyophilized cytohelicase (Sigma [Sigma Chemical], St. Louis, MO) and 3 mg of desalted, lyophilized pectinase (Sigma) were added. The anthers were then bisected transversely to their long axes with a microscalpel. After 45 min of incubation, the contents of several anthers were squeezed out with steel dissecting needles. Living protoplasts in ≤ 0.5 µl of digestion medium were drawn (aspirated) into a siliconized micropipette and gently blown out into 10 µl of bursting medium [aqueous 0.05%, octylphenoxy polyethoxyethanol (IGEPAL, Sigma) that was 0.01% potassium dextran sulfate and 0.04% paraformaldehyde, pH 8.5] suspended at the tip of a plastic pipette. This mixture was touched-off onto the middle of a wiped, glow-discharged glass slide where the suspension spread over the surface. A further 10 μl of bursting medium were added. After 10–20 sec, the slide was taken to a hood, and the slide's surface was immediately given 30 passes of nebulized aqueous 4% paraformaldehyde (pH 8.5). Then the slide was air-dried in the hood for 1 hr. Slides were passed through three washes consisting first of 10 sec in 140 ml of an aqueous 0.4% solution of Photo-Flo 200 with a drop of aqueous 0.05 M sodium borate added, followed by two 10 sec washes in 140 ml of distilled water. Slides were air-dried for 1 hr before storage at −80° in sealed plastic slide boxes.
Young shoot tissue (100 mg) was diced into ∼0.5 cm fragments, placed in a 1.7 ml microtube containing 500 µl of freshly prepared extraction buffer (200 mM Tris-HCl, pH 7.5; 25 mM ethylenediaminetetraacetic acid [EDTA], pH 8.0; 250 mM NaCl; 0.5% SDS; and 5 mM dithiothreitol), and stored at −80°. Subsequently, the suspension was heated to 65° for 10 min and then gently macerated for 15–20 sec in the microtube using a disposable pestle. The suspension was centrifuged at maximum speed (16,000 rpm) in a microcentrifuge at room temperature for 10 min. The supernatant was mixed with an equal volume of isopropanol to precipitate DNA. The suspension was again centrifuged at maximum speed at room temperature. The supernatant was removed, and the pellet was cleaned with 70% ethanol and dried. The dried pellet was suspended in 100 µl of sterile deionized water and treated with 100 µg/ml RNase A for 5 min at room temperature before the DNA was precipitated with 70% ethanol at −20° for 10 min. After centrifugation, the pellet was resuspended in 200 µl of deionized water. The DNA was quantified and adjusted to a final concentration of 100 ng/µl.
The 180-bp knob repeat was amplified and labeled according the manufacturer's instructions using the Roche PCR DIG Labeling Mix, maize genomic DNA, and appropriate primers (F: 5ʹ-CAACGCCCATTTTTATCGAA-3ʹ, R: 5ʹ-CGACCAGAGGATCGTACACC-3ʹ) to yield a 164-bp digoxygenin-labeled PCR product. The probe was purified according to the manufacturer's instructions using a Marligen PCR cleanup kit. SC spreads on glass microscope slides were treated in one of two ways (see below).
Air-dried SC spreads on slides were fixed for 10 min in aqueous 4% paraformaldehyde pH 8.5 and then passed through three washes, consisting first of 10 sec in 140 ml of an aqueous 0.4% solution of Photo-Flo 200 to which one drop of aqueous 0.05 M sodium borate was added, followed by two 10 sec washes in 140 ml of distilled water. Slides were air-dried overnight at room temperature. Then a ∼25 × 50 mm rectangle of Tetko nylon screen was placed over the SC spreads, and two drops of aqueous 33% silver nitrate was added. The slide was placed in a Y-Petri dish so it was resting above 5 ml of water in the bottom of the dish. The petri dish was closed and floated in a closed water bath at 40° until the nylon screen turned light brown (∼25 min). The nylon screen was gently washed off with distilled water, and the slide was allowed to air-dry.
Air-dried SC spreads on slides were immunolabeled using the procedure described by Lohmiller et al. (2008) with affinity-purified rat antibodies to maize (Zea mays) AFD1 protein diluted 1:50 and affinity-purified rabbit antibodies to tomato (Solanum lycopersicum) MLH1 protein diluted 1:50, followed by goat anti-rat 549 and goat anti-rabbit 488 (both from Jackson ImmunoResearch Laboratories), respectively, each at 1:500 dilution. Primary antibody incubations were done overnight at 4°, and secondary antibody incubations were done for 2 hr at 37°. The slides were stained with aqueous DAPI (10 µg/ml), washed with Tris-buffered saline (TBS) containing 0.05% Triton X, and cover glasses were mounted with Vectashield antifade mounting medium (Vector Laboratories). Slides were imaged before FISH, as described below.
FISH was performed as described (Zhong et al. 1996; Chang et al. 2007) on pachytene chromosome squashes, on SC spreads that were silver-stained and previously imaged, and on SC spreads that were immunostained with AFD1 and MLH1 and previously imaged. The cover glass and Vectashield mountant was removed from immunostained slides in deionized water, and then the slides were air-dried. For FISH, slides were incubated for 1 min in 45% v/v acetic acid, fixed with 1:3 acetic ethanol for 1 min, washed briefly in deionized water, and then digested with 100 µg/ml aqueous RNase A for 5 min at room temperature followed by pepsin (5 µg/ml in 0.01 M HCl) for 8 min at 37°. After briefly rinsing the slides in deionized water followed by 2 × SSC, the slides were fixed for 10 min in 1% w/v paraformaldehyde at pH 8.5, washed three times for 3 min each time in 2 × SCC, briefly rinsed in deionized water, dehydrated in 100% ethanol for 3 min at room temperature, and air-dried. Then, 20 µl of hybridization mixture [aqueous 2 × SSC containing 100 ng of labeled probe, 50% (v/v) formamide, 10% (w/v) sodium dextran sulfate, and 0.25% (w/v) sodium dodecyl sulfate] was placed on the slide, and a cover glass was added. Slides were heated for 2.5 min on an aluminum block at 80° to denature the DNA, and then slides were incubated at 37° in a moist chamber for at least 12 hr to permit hybridization. Slides were washed three times in aqueous 2 × SSC that was 50% v/v formamide at 42° for 80% stringency (Schwarzacher and Heslop-Harrison 2000). Slides were labeled with sheep anti-digoxygenin (Roche) followed by donkey anti-sheep conjugated to tetra-methyl rhodamine isothiocyanate (TRITC) (Jackson ImmunoResearch Laboratories) diluted 1:125 and 1:100, respectively, in 5% aqueous Roche blocking reagent. Antibody incubations were performed at 37° in 1-hr increments with three 3-min washes in an aqueous solution that was 100 mM Tris, 150 mM NaCl, and 0.05% v/v Tween-20, pH 7.5, after each incubation step. Donkey serum (5%) was added to the blocking buffer during the second secondary antibody incubation. After immunolabeling, slides were dehydrated through an ethanol series and air-dried. Cover glasses were mounted with Vectashield (Vector Laboratories) containing DAPI at 5 µg/ml.
Images of partial and complete SC sets were captured using IP Lab software (version 4) using a 100 × 1.4 numerical aperture (NA) apochromatic objective and a cooled Hamamatsu monochrome 1344X1044 pixel camera attached to a Leica DM 5500B microscope with a Prior motorized stage. The microscope was equipped for bright field, phase contrast, and fluorescence microscopy, with zero pixel shift filter cubes for DAPI, FITC, and TRITC.
The stage coordinates of individual nuclei were recorded so that the same nucleus could be imaged sequentially after air-drying, silver staining, fluorescence immunolabeling, and/or FISH, as appropriate. Images of the same nuclei were overlaid using CS2 Adobe Photoshop, but the layers were kept separate for later analysis. For each interstitial knob, the MLH1 layer alone was also examined to visualize and mark even dim MLH1 loci that are often difficult, if not impossible, to see in merged images. Bitmap images of the merged layers of the same nuclei were used to make measurements of SC lengths and mark the positions of kinetochores, knobs, and MLH1 foci using MicroMeasure (http://rydberg.biology.colostate.edu/MicroMeasure).
Complete imaging data sets and reagents are available upon request.
Using traditional pachytene squashes, pericentric heterochromatin and knobs in distal euchromatin are visible by both phase contrast microscopy and by fluorescence microscopy after staining DNA with DAPI (Figure 3, A and B). FISH using the 180-bp knob repeat demonstrated that the probe hybridized selectively to all five of the knobs observed in KYS primary microsporocytes (Figure 3C). Three of the knobs are located interstitially in the long arms of chromosomes 5, 6, and 7, and two of the knobs are located terminally on the short arms of chromosomes 1 and 9 (McClintock et al. 1981; Kato et al. 2004).
KYS maize pachytene chromosome squash. (A) By phase microscopy, large interstitial knobs (arrowheads) on the unstained chromosomes are visible along with smaller terminal knobs (small arrows). Pericentric heterochromatin is visible as darker segments of bivalents that are often about one-third of the length of each bivalent, e.g., observe the bivalent at the lower center. (B) The same squash after DAPI staining. Knobs are prominent bright blue enlargements along bivalents indicating a high concentration of DNA in knobs. Pericentric heterochromatin is also brighter than the distal euchromatin indicating higher concentrations of DNA in pericentric heterochromatin (but less than in knobs). (C) The same squash after FISH with the 180-bp knob repeat (red). Large interstitial knobs (arrowheads) and the smaller terminal knobs (small arrows) are specifically labeled with the 180-bp repeat probe. Bar, 5 µm.
We first tested whether maize knobs are attached to segments of SC that are short compared to the length of their corresponding knobs, as proposed in our model (Figure 2). For this, maize SC spreads were stained with silver to reveal SCs as long, dark threads with distinct kinetochores (Figure 4A). This permitted each of the 10 maize SCs to be identified on the basis of its relative length and arm ratio (Anderson et al. 2003). Next, DAPI staining showed a diffuse blue sheath around each SC as a result of the hypotonic dispersion of chromatin loops (Figure 4B). Finally, FISH using the 180-bp knob repeat showed numerous loops of knob chromatin (red) extending far from their distinct attachment sites on the SCs. At least some of these knob loops are a great deal longer than the blue-stained chromatin loops extending from the nonknob parts of the SCs (Figure 4, C and D). The interstitial knobs on chromosomes 5, 6, and 7 consisted of multiple loops of chromatin attached along well-defined segments of SC that averaged 1.7, 0.8, and 1.7 µm in length, respectively (Table 1). Measurements of interstitial knobs on traditional squashed pachytene chromosomes stained with DAPI or aceto-orcein were similar in length (0.5–2.0 µm). Thus, the length of SC occupied by these interstitial knobs in SC spreads is similar to the length of the same knob observed in a squash. We could not make similar comparisons for the terminal knobs on SCs 1 and 9 because the loops appeared to extend from the ends of these SCs (Figure 4, C and D).
Maize pachytene SC spread. (A) SCs and kinetochores stained with silver. Each SC has been identified on the basis of relative length and arm ratio, and numbered at its kinetochore. Kinetochores on SCs 1 and 7 are stuck together as are the kinetochores on SCs 2 and 5. SC 8 is partially asynapsed. (B) The same SC spread visualized with DAPI fluorescence. Due to the hypotonic spreading procedure, the chromatin loops around the SCs are dispersed laterally as a fuzzy blue coat along the length of each SC. (C) The FISH signal (red) of the 180-bp repeat for this SC spread. (D) Merged images of silver-stained SCs (inverted to appear white), DAPI-stained chromatin (blue), and the 180-bp knob repeat (red). The chromatin of the interstitial knobs (on the long arms of SCs 5, 6, and 7) occupies well-defined SC segments, and at least some chromatin loops from the knobs (red) extend much farther from their SC attachment sites than chromatin loops in distal euchromatin and pericentric heterochromatin. For the terminal knobs (on the short arms of SCs 1 and 9) the loops also extend far from their attachment sites, but the loops appear to extend from the tips of the SCs without defining distinct terminal SC segments. Bar, 5 µm. SC, synaptonemal complex.
Next, we examined spread maize SCs that had been labeled with antibodies to maize AFD1 protein and crossover sites on these SCs that had been labeled with antibodies to tomato MLH1 protein (Golubovskaya et al. 2006; Lhuissier et al. 2007). Most SCs have one or two MLH1 foci, and we found an average of 13.9 MLH1 foci per SC set (n = 18) (Figure 5). Individual MLH1 foci varied in brightness, possibly depending on the amount of the MLH1 protein present in individual RNs (Anderson et al. 2014). Using the 180-bp knob repeat for FISH on these previously immunolabeled SC spreads allowed us to compare the location of knobs and MLH1 fluorescent foci simultaneously on the same SCs (Figure 5). Unfortunately, the immunolocalization procedure for AFD1 and MLH1 that preceded FISH blurred the edges of knobs compared with their appearance without the immunolocalization procedure (compare Figure 4, C and D to Figure 5). Even so, we observed MLH1 foci near and apparently within interstitial knobs on the long arms of SCs 5, 6, and 7. We observed a total of 255 SCs with interstitial knobs (Table 2) and MLH1 foci. Of the total 367 MLH1 foci observed on these SCs, 25 MLH1 foci were near a knob (defined as within 1 µm to either side of a knob) and 16 MLH1 foci were within a knob. Using data on average SC lengths plus the average SC length occupied by the knobs on these chromosomes (Table 1), we calculated frequencies of MLH1 foci/μm for whole SCs, in knobs, and near knobs (1 µm in euchromatin to either side of the knob = 2 µm SC length for each SC) (Table 2). We found similar frequencies of MLH1 foci/μm for each SC (0.042–0.046). For MLH1 foci in knobs, frequencies ranged from 0.026 to 0.056 µm−1, and for MLH1 foci near knobs frequencies ranged from 0.039 to 0.056 µm−1 (Table 2). While the frequencies of MLH1 foci within and near knobs for SCs 6 and 7 are close (0.052 vs. 0.056 µm−1 and 0.056 vs. 0.053 µm−1, respectively), the frequencies of RNs in and near the knob on SC 5 are lower (0.026 and 0.039 µm−1, respectively.
Portions of maize SC spreads labeled with antibodies to MLH1 (white foci) and AFD1 (red, to show the SC axes) and hybridized to the 180-bp knob repeat (blue). MLH1 foci are observed near (arrow) and in (arrowheads) knobs. (A) Portion of a pachytene SC spread showing an MLH1 focus near the edge of the knob on SC 5. Other MLH1 foci in this image are not in or near knobs. (B) Portion of an SC spread in early diplotene showing an MLH1 focus in the interstitial knob of the long arm of SC 6. (C) Portion of a pachytene SC spread showing an MLH1 focus in the interstitial knob on the long arm of SC 7. (D) Portion of a pachytene SC spread showing an MLH1 focus in the interstitial knob of SC 5 and a small MLH1 focus at the tip of the short arm of SC 9 (small arrowhead) where the terminal knob is located. Bar, 5 µm. SC, synaptonemal complexes.
In addition, we observed 92 MLH1 foci on 52 SC 1s and found only one MLH1 focus at the site of the small terminal knob, and we observed 94 MLH1 foci on 73 SC 9s and found three MLH1 foci at the site of SC 9's larger terminal knob (Figure 5D). Because we were unable to measure the length of SC in these terminal knobs, we were also unable to determine frequencies of MLH1 foci/μm of SC for these knobs.
Our earlier observations of RNs on KYS maize SCs indicated that crossing over occurs in knob heterochromatin at frequencies similar to the surrounding euchromatin (Figure 1) (Anderson et al. 2003). However, this work had the potential problem that knobs were not observed directly on the SC spreads used for RN mapping, so the exact location and size of knobs had to be inferred from other data. However, even if the projected knob locations on SCs were not precisely correct, it was clear that there were no 0.5–2.0 µm segments of SCs near knob sites that lacked RNs, as would be expected if knob heterochromatin blocked meiotic crossing over. Therefore, we offered a model for knob structure that would make our observations compatible with the assumption that crossing over is suppressed in heterochromatin. In this model, a knob consists of a few long loops of condensed chromatin, which have a tiny site of insertion on the SC compared to the size of the knob (Figure 2).
In the research described here, we used FISH of the maize 180-bp knob repeat to determine the location of knobs on SC spreads and the length of SC into which loops of knob heterochromatin were inserted. In agreement with our model, DAPI staining and FISH showed that many (possibly all) of the hypotonically dispersed loops of knob chromatin are much longer than typical hypotonically dispersed nonknob chromatin loops (Figure 4). However, in disagreement with our model, chromatin loops of the interstitial knobs on SCs 5, 6 and 7 did not have tiny insertion sites but were inserted all along well-defined SC segments that were similar in length to the corresponding knobs measured on squashed pachytene chromosomes (Results and Table 1). The observation that knob chromatin occupies a significant segment of SC length is also consistent with earlier reports that knobs increase the length of pachytene chromosomes by the length of the knobs, and with reports that knobs buckle out in heterozygotes, presumably due to the different lengths of SC lateral elements (Longley 1937; Dempsey 1994). Thus, our earlier report (Anderson et al. 2003) that RNs occur at presumed heterochromatic knob sites in maize together with our current observation that loops of knob chromatin are inserted all along the length of interstitial knobs strongly suggests that meiotic crossing over occurs regularly within these knobs.
We also used immunofluorescent localization of AFD1 protein and MLH1 protein, and FISH localization of the 180-bp knob repeat, to directly observe the locations of MLH1 foci and knobs on the same SCs. All of the interstitial knobs on SCs 5, 6, and 7 occur within distal euchromatin. The frequency of MLH1 foci/μm was essentially the same in and to either side of the knobs on SCs 6 and 7 (0.052–0.056 MLH1 foci/μm of SC), indicating that these knobs have no effect on the rate of crossing along the SC (Table 2). For SC 5, the frequency of MLH1 foci/μm of SC is lower in the knob than to either side of the knob (0.026 vs. 0.039 MLH1 foci/μm of SC, respectively), and both of these values are lower than the frequencies for the knobs on SCs 6 and 7. However, because of the small sample sizes, it is unclear whether these values represent real differences between the knobs, but in support of a difference, there is a dip in RN frequency at the knob site on SC 5 in Figure 1. From the data, we conclude that crossing over occurs in all of these knobs, and the knobs either have no or only a modest effect on the frequency of crossing over along SC nearby or within their boundaries.
Crossovers visualized by MLH1 foci represent type I crossovers, i.e., those that show interference (Mercier et al. 2015). In maize, mathematical modeling indicated that about 80% of the crossovers as assessed by RNs are type I crossovers with the remaining RNs (not labeled by MLH1) being type II crossovers, i.e., those that do not show interference (Falque et al. 2009; Mercier et al. 2015). Based on these results and an average of about 20 RNs per nucleus (Anderson et al. 2003), one would expect an average of about (0.8 × 20) 16 MLH1 foci per nucleus in maize. Instead, we observed an average of about 14 MLH1 foci per nucleus. While it is not clear why fewer MLH1 foci were observed than expected, possible explanations include transient detection of MLH1 foci as observed for mice (Anderson et al. 1999), a lower efficiency of immunolabeling (perhaps caused by using antibodies raised against tomato MLH1 protein), and/or an error in modeling. In any case, the lower than expected number of MLH1 foci could indicate that we may be underestimating the total number of type I crossovers. In addition, type II crossovers that may comprise 20% of all crossovers are not detected by MLH1 antibodies, so the total amount of crossing over in and near knobs may be ∼20% higher than we observed based on MLH1 foci (Table 2). Together, these results indicate that the reported frequencies of MLH1 foci in and around knobs are probably minimal estimates of crossing over, and that the actual amount of crossing over in these locations may be substantially higher. Furthermore, in tomato, type I and type II crossovers have different distributions, with type II crossovers being observed more often than expected in the short arms of acrocentric chromosomes and in pericentric heterochromatin compared to type I crossovers (Anderson et al. 2014). If the same pattern applies to maize, it could explain why only a few MLH1 foci were observed at the ends of the terminal knob-carrying short arms of SC 1 and SC 9, with most of the many RNs observed at those positions presumably representing type II crossovers (Figure 1) (Anderson et al. 2003).
We have shown that the rate of crossing over per unit length of SC is not suppressed in knob heterochromatin compared to euchromatin. On the other hand, chromatin loops in knobs are much longer than chromatin loops in euchromatin, and assuming that the chromatin loops occur at the same frequency per micrometer of SC in euchromatin and heterochromatin (Zickler and Kleckner 1999), there must be more DNA per micrometer of SC in knobs compared to SC in euchromatin. If so, there must be proportionately less crossing over per unit length (base pairs) of DNA in knobs compared to nearby euchromatin. A similar effect of DNA loop length on crossover frequency was suggested earlier based on observations in other species (Stack 1984). To date, no accurate measurements have been made of the amount of DNA in maize knobs. The most similar comparison comes from the work of Peterson et al. (1996), who used Feulgen staining and microdensitometry of tomato pachytene chromosomes to estimate that there is at least fivefold more DNA per micrometer of SC in pericentric heterochromatin compared to distal euchromatin. If this estimate is applied to maize knobs, the crossover rate per base pair of knob DNA would be only one-fifth the crossover rate per base pair of DNA in nearby euchromatin. This ratio of crossovers is similar to that reported for euchromatin compared to knob-like heterochromatin in Arabidopsis thaliana (The Cold Spring Harbor Laboratory, Washington University Genome Sequencing Center, and PE Biosystems Arabidopsis Sequencing Consortium 2000; Fransz et al. 2000). Thus, it seems that whether or not crossing over is suppressed in knobs compared to euchromatin depends on the context: crossing over is suppressed in the context of DNA amount but not suppressed in the context of SC length.
Knob heterochromatin has been reported to influence recombination and preferential segregation, but no genes have been localized in maize knobs, and for that reason knobs do not appear on linkage maps (Rhoades 1978; Ananiev et al. 1998b; Ghaffari et al. 2013). Recently, Ghaffari et al. (2013) were able to map three knobs in gene-rich regions onto the reference genome for maize B73. Ghaffari et al. (2013) observed a reduction in crossing over in markers closely linked to a small knob in the homozygous condition, although without accurate estimates of the amount of DNA in the knob they were unable to quantify the reduction on a centiMorgan per megabase (cM/Mb) scale. For knobs in the heterozygous condition (knob/knobless), they found as much as a twofold reduction in euchromatic crossing over in cM/Mb near knob sites. However, given previous evidence that heterozygous knobs often cause local asynapsis, this reduction of crossing over is more likely due to interference with synapsis than to the effect of knob heterochromatin per se (Rhoades and Dempsey 1966; Rhoades 1978; Dempsey 1994; Fransz et al. 2000). So far, there is no molecular evidence for crossing over in knobs, but the variability in the sizes of knobs within maize populations (Kato 1976), "suggests recombination and unequal crossing over is frequent within knobs…" (Buckler IV et al. 1999). More generally, changes in the number of tandem repeats, such as those found in knobs, are often explained by unequal crossing over, although such changes would not necessarily have to occur during meiosis (Smith 1976; Yamamoto and Miklos 1978). Recent sequencing of the special centromeric chromatin in the B73 maize chromosome 10 indicates that it has been involved in many double-strand break-induced rearrangements (including inversions that have moved three genes into this centromere). However, because the rearrangements appear to be mediated by microhomology that typically involves ∼5 bp overlaps, it is unlikely that they are due to homologous meiotic recombination that involves matching sequences hundreds of base pairs long (Paques and Haber 1999; Wolfgruber et al. 2016). With further improvements in sequencing difficult genomic regions, it may become possible to determine whether similar types of rearrangements also occur in knob heterochromatin and whether they are mediated by meiotic homologous recombination.
Our observations of RNs at knob sites (Anderson et al. 2003) and of MLH1 foci in knobs (this work) do not necessarily mean that crossing over is occurring in the 180-bp tandem repeats that comprise the bulk of DNA in most maize knobs. Other sequences have been observed in knobs, including retrotransposons and 350-bp TR-1 element repeats that are related to the 180-bp repeats (Ananiev et al. 1998a; Ghaffari et al. 2013; Wolfgruber et al. 2016). In addition, maize genome assemblies are not good enough to rule out the presence of other interspersed low copy sequences scattered among 180-bp knob repeats. Thus, our results indicate only that crossing over is occurring within knob heterochromatin, but not which sequences are involved in crossing over or in attachment of knob loops to SC lateral elements.
Although there is no doubt that crossing over is suppressed in some heterochromatin, it has not been clear whether all heterochromatin suppresses crossing over. Considering the multiple factors involved in crossing over (Ellermeier et al. 2010; Vader et al. 2011; Vincenten et al. 2015) and that there are different types of heterochromatin based on behavior (constitutive vs. facultative), staining characteristics, degrees of condensation, chemical modifications, and various constituents and locations (Loidl 1987; Carlson 1988; Craig 2004; Roudier et al. 2011; van Steensel 2011), it should not be surprising that different heterochromatins could vary in the characteristic of meiotic crossing over. Based on our observations of RNs and MLH1 foci in maize knobs, it seems likely that crossing over occurs at a similar rate per unit length of SC within knobs and in euchromatin to either side of knobs. However, because there is more DNA per unit length of SC within a knob compared to SC in adjacent euchromatin, there is proportionately less crossing over per base pair within knobs compared to adjacent euchromatin (Gaut et al. 2007). Thus, crossing over in knob heterochromatin is not suppressed in the context of SC length, while crossing over is suppressed, but not eliminated, in the context of DNA length.
We thank Zac Cande for the antibodies to maize AFD1 protein and Song-Bin Chang for selecting the primers for PCR amplification of the 180-bp maize knob repeat sequence.
, 1988 Synaptonemal complex spreading in Allium cepa and Allium fistulosum. II. Pachytene observations: the SC karyotype and the correspondence of late recombination nodules and chiasmata. Genome 30: 399–410.
, 1998a Complex structure of the knob DNA on maize chromosome 9: retrotransposon invasion into heterochromatin. Genetics 149: 2025–2037.
, 1998b A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: are chromosome knobs megatransposons? Proc. Natl. Acad. Sci. USA 95: 10785–10790.
, 2003 High resolution crossover maps for each bivalent of Zea mays using recombination nodules. Genetics 165: 849–865.
, 2014 Combined fluorescent and electron microscopic imaging unveils the specific properties of two classes of meiotic crossovers. Proc. Natl. Acad. Sci. USA 111: 13415–13420.
, 1958 Crossing over in heterochromatin. Am. Nat. 92: 59–60.
, 2000 The many hues of plant heterochromatin. Genome Biol. 1: 107.1–107.4.
, 1991 C-band and chiasma distribution in Scilla siberica (Hyacinthaceae). Genome 34: 179–189.
, 2007 Mapping of meiotic single-stranded DNA reveals double-strand-break hotspots near centromeres and telomeres. Curr. Biol. 17: 2003–2012.
, 1949 The structure and meiotic behavior of the differentiated chromosomes of tomato. Genetics 34: 437–461.
, 1988 The cytogenetics of corn, pp. 259–331 in Corn and Corn Improvement, edited by Sprague G. F., Dudley J. W.. Crop Science Society, Madison, WI.
, 1975 Electron microscopy of meiosis in Drosophila melanogaster females: II: the recombination nodule - a recombination-associated structure at pachytene? Proc. Natl. Acad. Sci. USA 72: 3186–3189.
, 1999 AFLP markers in a molecular linkage map of maize: codominant scoring and linkage group distribution. Theor. Appl. Genet. 99: 425–431.
, 1984 Evidence of nonchiasmate bonds at metaphase I in inbred rye. Can. J. Genet. Cytol. 26: 409–414.
, 2007 Predicting and testing physical locations of genetically mapped loci on tomato pachytene chromosome 1. Genetics 176: 2131–2138.
, 2000 The pachytene chromosomes of maize as revealed by fluorescence in situ hybridization with repetitive DNA sequences. Theor. Appl. Genet. 101: 30–36.
, 1998 Why is the centromere so cold? Genet. Res. 8: 81–82.
, 1994 The relationship between genetic and physical distances in the cloned al-sh2 interval of Zea mays L. genome. Proc. Natl. Acad. Sci. USA 91: 8268–8272.
, 1972 The structure and function of chromatin, pp. 237–431 in Advances in Human Genetics, edited by Harris H., Hirschhorn K.. Plenum Press, New York.
, 2005 Heterochromatin–many flavours, common themes. Bioessays 27: 17–28.
, 1989 Meiosis in trisomic female mice with Robertsonian translocations II. Chromosome behavior at first and second meiotic prophases. Cytogenet. Cell Genet. 52: 118–123.
, 1937 Recent Advances in Cytology. The Blakiston Company, Philadelphia.
, 1986 Heterochromatin readjusting chiasma distribution in two species of the genus Arcyptera: the effect among individuals and populations. Heredity 56: 177–184.
, 1994 Traditional analysis of maize pachytene chromosomes, pp. 432–441 in The Maize Handbook, edited by Freeling M., Walbot V.. Springer-Verlag, New York.
, 1974 C-banding and non-homologous associations in Gryllus argentinus. Chromosoma 48: 367–390.
, 2002 Chromosomal rearrangements and evolution of recombination: comparison of chiasma distribution patterns in standard and Robertsonian populations of the house mouse. Genetics 162: 1355–1366.
, 2009 Two types of meiotic crossovers coexist in maize. Plant Cell 21: 3915–3925.
, 1974 Heterochromatin content and chiasma distribution in the megameric chromosomes of Stethophyma gracile and S. lineatum (Orthoptera: Acrididae). Chromosoma 46: 375–395.
, 2002 Male mouse recombination maps for each autosome identified by chromosome painting. Am. J. Hum. Genet. 71: 1353–1368.
, 2013 Maize chromosomal knobs are located in gene-dense areas and suppress local recombination. Chromosoma 122: 67–75.
, 2016 Heterochromatin-associated proteins HP1a and Piwi collaborate to maintain the association of achiasmate homologs in Drosophila oocytes. Genetics 203: 173–189.
, 1975 Heterochromatic connectives between the chromosomes of Secale cereale. Can. J. Genet. Cytol. 17: 269–273.
, 1976 Homologous and non-homologous chromosome associations by interchromosomal chromatin connectives in Ornithogalum virens. Chromosoma 57: 309–318.
, 1966 The effects of autosomal euchromatin on crossing over in the X chromosome of Drosophila melanogaster. Am. Nat. 100: 159–162.
, 1928 Das heterochromatin der Moose. I. Jahrb. Wiss. Botanik. 69: 762–818.
, 1971 Distribution of constitutive heterochromatin in mammalian chromosomes. Chromosoma 34: 243–253.
, 2002 A molecular cytogenetic map of sorghum chromosome 1: fluorescence in situ hybridization analysis with mapped bacterial artificial chromosomes. Genetics 161: 345–353.
, 1976 Myths and mechanisms of meiosis. Chromosoma 54: 295–325.
, 1965 The meiotic system. Protoplasmatologia 6: 1–335.
, 1980 Heterochromatin variation in Cryptobothrus chrysophorus III. Synthetic hybrids. Chromosoma 78: 165–186.
, 1985 Pseudoterminalization, terminalization, and non-chiasmate modes of terminal association. Chromosoma 92: 89–99.
, 1978 Giemsa C-banding of rye meiotic chromosomes and the nature of "terminal" chiasmata. Chromosoma 66: 45–57.
, 1987 Chiasmata, pp. 213–244 in Meiosis, edited by Moens P. B.. Academic Press, New York.
, 1976 Cytological studies of maize [Zea Mays L.] and Teosinte [Zea Mexicana Schrader Kuntze] in relation to their origin and evolution. Mass. Agr. Exper. Stat. Bul. 635: 1–185.
, 2005 Comprehensive molecular cytogenetic analysis of sorghum genome architecture: distribution of euchromatin, heterochromatin, genes and recombination in comparison to rice. Genetics 171: 1963–1976.
, 1974 Heterochromatin distribution and chiasma localization in the grasshopper Bryodema tuberculata (Fabr.) (Acrididae). Chromosoma 44: 393–404.
, 1993 A cytogenetically based physical map of chromosome 1B in common wheat. Genome 36: 467–475.
, 1986 Repression of meiotic crossing over by a centromere (CEN3) in Saccharomyces cerevisiae. Genetics 114: 769–789.
, 1990 Visualization of DNA in pachytene by monoclonal antibodies against BrdU reveals synaptonemal complex-like structures. Cytogenet. Cell Genet. 54: 24–28.
, 1933 Cytological studies in Allium. IV. Allium fistulosum. Sven. Bot. Tidskr. 27: 211–232.
, 2007 The mismatch repair protein MLH1 marks a subset of strongly interfering crossovers in tomato. Plant Cell 19: 862–876.
, 2008 Meiotic chromatin: the substrate for recombination initiation, pp. 165–193 in Recombination and Meiosis: Models, Means and Evolution, edited by Egel R., Lankenau D. H.. Springer-Verlag, Berlin.
, 1955 Die struktur der pachytanchromosomen in euchromatin und heterochromatin und ihre auswirkung auf die chiasmabildung bei salvia-arten. Chromosoma 7: 90–128.
, 2008 Cytological analysis of MRE11 protein during early meiotic prophase I in Arabidopsis and tomato. Chromosoma 117: 277–288.
, 1979 C-band proximity of chiasmata and absence of terminalisation in Allium flavum (Liliaceae). Chromosoma 73: 45–51.
, 1987 Heterochromatin and differential chromosome staining in meiosis. Biol. Zent. 106: 641–662.
, 1937 Morphological characters of teosinte chromosomes. J. Agric. Res. 54: 835–862.
, 2003 Mlh1p and Mlh3p localize to precociously induced chiasmata of okadaic acid treated mouse spermatocytes. Genetics 165: 2283–2287.
, 1933 The relation between chiasmata and crossing over in diploid and triploid Drosophila melanogaster. J. Genet. 27: 343–360.
, 1939 Crossing over and heterochromatin in the X chromosome of Drosophila melanogaster. Genetics 24: 413–435.
, 1981 Chromosome Constitution of Races of Maize. Colegio de Postgraduados, Chapingo, Mexico.
, 1976 Telomeric satellite DNA functions in regulating recombination. Chromosoma 56: 143–167.
, 1971 The meiotic behaviour of autosomal heterochromatic segments in hedgehogs. Chromosoma 35: 143–152.
, 2005 Ribosomal DNA heterochromatin in plants. Cytogenet. Genome Res. 109: 104–111.
, 1981 Highly repeated DNA squence limited to knob heterochromatin in maize. Proc. Natl. Acad. Sci. USA 78: 4490–4494.
, 1996 DNA content of heterochromatin and euchromatin in tomato (Lycopersicon esculentum) pachytene chromosomes. Genome 39: 77–82.
, 1977 Simple Mendelian inheritance of the reiterated ribosomal DNA of yeast. Proc. Natl. Acad. Sci. USA 74: 5091–5095.
, 1967 Structure and homology between pachytene and somatic metaphase chromosomes of the tomato. Genetica 38: 115–133.
, 1987 Investigating the genetic control of biochemical events in meiotic recombination, pp. 157–210 in Meiosis, edited by Moens P. B.. Academic Press, Orlando.
, 1955 The cytogenetics of maize, pp. 123–219 in Corn and Corn Improvement, edited by Sprague G. F.. Academic Press, New York.
, 1978 Genetic effects of heterochromatin in maize, pp. 641–671 in Maize Breeding and Genetics, edited by Walden D. B.. John Wiley and Sons, New York.
, 1966 The effect of abnormal chromosome 10 on preferential segregation and crossing over in maize. Genetics 53: 989–1020.
, 1979 Chromosome structure and levels of chromosome organization. Cell Biol. 2: 267–361.
, 2000 Practical In Situ Hybridization. BIOS Scientific Publishers Ltd., New York.
, 1995 Two-dimensional spreads of synaptonemal complexes from solanaceous plants. VI. High-resolution recombination nodule map for tomato (Lycopersicon esculentum). Genetics 141: 683–708.
, 1976 Evolution of repeated DNA sequences by unequal crossover. Science 191: 528–535.
, 1984 Heterochromatin, the synaptonemal complex, and crossing over. J. Cell Sci. 71: 159–176.
, 1991 Staining plant cells with silver. II. Chromosome cores. Genome 34: 900–908.
, 1996 Localized chiasmata and meiotic nodules in the tetraploid onion Allium porrum. Genome 39: 770–783.
, 2009 Electron microscopic immunogold localization of recombination-related proteins in spreads of synaptonemal complexes from tomato microsporocytes, pp. 147–169 in Meiosis Volume 2 Cytological Methods, edited by Keeney S.. Humana Press, Inc., Totowa, NJ.
, 1990 Unequal crossing over and heterochromatin exchange in the X-Y bivalents of the deer mouse, Peromyscus beatae. Chromosoma 99: 183–189.
, 2003 Chromosomes: Organization and Function. Blackwell Science Ltd., Malden, MA.
, 1984 An analysis of regional constraints on exchange in Drosophila melanogaster using recombination-defective meiotic mutants. Genetics 106: 45–71.
The Cold Spring Harbor Laboratory, Washington University Genome Sequencing Center, and PE Biosystems Arabidopsis Sequencing Consortium, 2000 The complete sequence of a heterochromatic island from a higher eukaryote. Cell 100: 377–386.
, 2006 Reinterpreting pericentromeric heterochromatin. Curr. Opin. Plant Biol. 9: 647–653.
, 2011 Protection of repetitive DNA borders from self-induced meiotic instability. Nature 477: 115–121.
, 2006 Construction of a 10,000-marker ultradense genetic recombination map of potato: providing a framework for accelerated gene isolation and genomewide physical map. Genetics 173: 1075–1087.
, 2011 Chromatin: constructing the big picture. EMBO J. 30: 1885–1895.
, 2016 High quality maize centromere 10 sequence reveals evidence of frequent recombination events. Front. Plant Sci. 7: 308.
, 1978 Genetic studies on heterochromatin in Drosophila melanogaster and their implication for the functions of satellite DNA. Chromosoma 66: 71–98.
, 2002 Molecular characterization of meiotic recombination across the 140-kb multigenic a1-sh2 interval of maize. Proc. Natl. Acad. Sci. USA 99: 6157–6162.
, 2003 Toward a saturated molecular genetic linkage map for cultivated sunflower. Crop Sci. 43: 367–387.
, 1971 Heterochromatin, satellite DNA, and cell function. Science 174: 1200–1209.
, 1996 High-resolution mapping on pachytene chromosomes and extended DNA fibres by fluorescence in-situ hybridization. Plant Mol. Biol. Report. 14: 232–242.


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text:
Home › The Wire
RENSSELAER POLYTECHNIC INSTITUTE (RPI) ENROLLS WALTERS-STORYK DESIGN GROUP
WSDG Designs Fully Immersive Integrated Media Complex
handsonpr@aol.com ⋅ Sep 29, 2017
TROY, NY: Rensselaer Polytechnic Institute (RPI), the world's 3rd highest ranked research university, has commissioned WSDG Walters-Storyk Design Group to design and oversee the construction of a fully immersive audio/video/ 3D production, mixing and editing complex for their Troy, NY campus. The primary mission for this addition to Rensselaer's Media Arts, Science & Technology Department is to advance the creation, performance and production of 3D immersive audio and video content. "RPI was founded in 1824 for the application of science to the common purposes of life, and is recognized for its degree programs in engineering, computing, business management and information technology," states, Mary Simoni, RPI, Dean-School of Humanities, Arts & Social Sciences. "This new facility will be integral to our signature thrust, and will significantly extend connectivity to (and collaboration with)the university's Experimental and Performing Arts Center (EMPAC), and Cognitive Immersive Systems Lab (CISL)."
"Our assignment focused on the complete renovation of the existing 1700 sq. ft. space, and the design, construction supervision and systems integration of a 1000 sq. ft. Audio Recording/Production Studio, a cutting edge 300 sq. ft. Audio Control Room, a 100 sq. ft.Iso lab, 160 sq. ft. Video Control Room and a 90 sq. ft. AV Lab," reports WSDG Partner/Project Manager Joshua Morris. "With a 16' ceiling height, and sufficient space for ambitious audio and video programming creation, RPI's Immersive Production complex will provide students with a future-proofed experimentation environment. Special attention is being focused on the facility's variable acoustic properties, to compensate for the 'deader' tracking environment required by Immersive production techniques," Morris said.
"The complex will utilize 'dark' fiber optic connectivity previously installed between RPI's EMPAC and DCC buildings, to allow for seamless audio/video connectivity. Advantages will include use of the EMPAC Concert Hall as a sound stage with audio/video control in the DCC and, enable a live performance at the DCC to be viewed on multiple EMPAC screens," Morris adds.
"I first encountered WSDG's expertise in designing audio/video production facilities during my tenure at the University of Michigan-Ann Arbor," Dean Simoni adds. "The success of that project motivated me to recommend them to RPI. WSDG's credits include multifaceted installations for NYU, the Berklee College of Music (Boston and Valencia, Spain) and many other major universities. Rob Carney, Project Manager for Campus Planning & Facilities Design, Kim Osburn, Manager of Operations & Administrative Services for the School of Humanities, Arts & Social Sciences, and I have been consistently impressed with WSDG's thinking, creative design recommendations, attention to detail and follow through. We look forward to the timely completion of our new Immersive Production facility in January 2018."
"WSDG's history of developing production/teaching complexes for leading colleges and universities includes educational facilities for many major schools," concludes founding partner, John Storyk. "Our projects range from the Berklee College of Music (Boston, Mass.& Valencia, Spain) to NYU's Steinhardt / Dolan Music Technology Center in NYC, ESPM's Broadcast Teaching Center in Sao Paulo, Brazil, the University of Applied Sciences in Solothurn, Switzerland, and over twenty Art Institute Teaching Centers throughout the U.S. As an adjunct professor of acoustics and studio design I lecture frequently at the Berklee College of Music. We are honored to have been retained by
RPI to prepare their sstudents for 21st Century audio production careers."
RPI Gets Immersive
Walters-Storyk Design Group Europe Tapped For DJ Studio
Russ Berger Design Group Nominated For TEC Award for Design of Blade Studios
GC Pro and Malvicino Design Group Collaborate to Bring Audio Production Facilities to the Monterrey Institute of Technology and Higher Education
Rensselaer Polytechnic Institute Turns To GC Pro
Rensselaer Hires WSDG for a Fully Immersive Integrated Media Complex
AUDIBLE – LEADING AUDIOBOOK PRODUCER TAPS WSDG WALTERS-STORYK DESIGN GROUP FOR NEW STUDIOS
MUSIC Group Acquires TC Group


In preparation for filming the Immigrant, I came across this website. Although I have not had time to read all the stories yet, it seems to provide an honest insight into the experiences and challenges of people who immigrated to America.
I suspect as we begin interviewing people for the film, their stories may be similar to the ones on this website.
It's Sunday 3:36 PM — Sorry, the Office is closed.


"The Model X can do this ballet thing to the Trans-Siberian Orchestra".
The question that comes right up in our minds "why would you do that?", is brilliantly swept out of the way by Musk: "Seemed like fun." Yes, sometimes it's the shortest answer that makes our day. Their discussion about this cultural touch to the Teslas continues with Joe asking how, besides to all ongoing earth- and space projects including sending rockets into space and powering Australia, he would find the time to do that. "There were some engineers at Tesla that said 'what if we make this car dance and play music?' And I was like 'That sounds great, please do it. Let's try to get it done in time for christmas!', and we did.", Mr. Musk replies with a small but sparkling smile.
Also, can we have Cannibal Corpse, too? Please!


If that doesn't work, you will have to copy skplayer. If you use a 64-skplayer.exe version of Windows, you should also copy skplayer. Open Windows Start menu and select "Run".
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text:
Dance Language of the Honey Bee
Waggle dance definition:
Communication system is defined as connect or communicate with other people, i.e. means that "any sharing of information". Honey bees use special types of complex communication system in the form of a dance language to communicate or to inform members about the location of a food source. This special type of dance system is called "Honey bee dance".
Why do bees do waggle dance:
The bee which locates the food source at first is called the forager bee. It communicates with other bees about the location of the food source through the dance form which is called waggle dance.
How do honey bees communicate with each other:
The forager bee after discovering a suitable source of food returns back to the hive and perform a dance on the comb to inform in-mates about the food source. This honey bee dance is of two types-
1.	Round dance
2.	Waggle dance
Round dance:
What is the round dance :
For food at short distances from 20-200 meters, returning foragers perform the round dance. The dance involves a series of circles with reversals in direction every second or so i.e. they dance in a circular fashion.
What does a honey bee round dance tell the other bees:
Round dance conveys the information about the source of food only.
Waggle dance(bee communication dance):
For food at greater distances i.e. beyond 200 meters, the returning foragers perform the waggle dance.
In this dance form, they make a figure '8' with a straight portion in the middle of the figure. The forager waggles its body and emits sound burst during the straight or waggling run. The direction of relative to the sun is same as the direction of straight run relative to gravity.
The duration of the straight run increases with distance at the rate of about one complete waggle per 30 meters. The frequency of the waggling decreases in increase in distance.
The area that the dance occupies on the comb, the duration of each complete figure '8' cycle and the duration of the sound bursts all correlate with the distance the food source.
It is now known from various studies, researches and experiments that the main source of information is the direction and the duration of the straight or wagging run. Both round and wagging communicate information about distance and direction of the food source to the other bees. Thus the waggle dance of the forager bees inform not only about a food source but also about its direction and distance from the comb.
Bee Dance