Christine J. Farr

Mouse transferrin receptor 1 is the cell entry receptor for mouse mammary tumor virus

Enveloped viruses enter cells by binding to their entry receptors and fusing with the membrane at the cell surface or after trafficking through acidic endosomal compartments. Species-specific virus tropism is usually determined by these entry receptors. Because mouse mammary tumor virus (MMTV) is unable to infect Chinese hamster cells, we used phenotypic screening of the T31 mouse/hamster radiation hybrid panel to map the MMTV cell entry receptor gene and subsequently found that it is transferrin receptor 1. MMTV-resistant human cells that expressed mouse transferrin receptor 1 became susceptible to MMTV infection, and treatment of mouse cells with a monoclonal antibody that down-regulated cell surface expression of the receptor blocked infection. MMTV, like vesicular stomatitis virus, depended on acid pH for infection. MMTV may use transferrin receptor 1, a membrane protein that is endocytosed via clathrin-coated pits and traffics through the acidic endosomes, to rapidly get to a compartment where acid pH triggers the conformational changes in envelope protein required for membrane fusion.

Generation of a human X-derived minichromosome using telomere-associated chromosome fragmentation

A linear mammalian artificial chromosome vector will require at least three functional elements: a centromere, two telomeres and replication origins. One route to generate such a vector is by the fragmentation of an existing chromosome. We have previously described the use of cloned telomeric DNA to generate and stably rescue truncated derivatives of a human X chromosome in a somatic cell hybrid. Further rounds of telomere‐associated chromosome fragmentation have now been used to engineer a human X‐derived minichromosome. This minichromosome is estimated to be < 10 Mb in size. In situ hybridization and molecular analysis reveal that the minichromosome has a linear structure, with two introduced telomere constructs flanking a 2.5 Mb alpha‐satellite array. The highly truncated chromosome also retains some chromosome‐specific DNA, originating from Xp11.21. There is no significant change in the mitotic stability of the minichromosome as compared with the X chromosome from which it was derived.

A bovine whole-genome radiation hybrid panel and outline map

A 3000-rad radiation hybrid panel was constructed for cattle and used to build outline RH maps for all 29 autosomes and the X and Y chromosomes. These outline maps contain about 1200 markers, most of which are anonymous microsatellite loci. Comparisons between the RH chromosome maps, other published RH maps, and linkage maps allow regions of chromosomes that are poorly mapped or that have sparse marker coverage to be identified. In some cases, mapping ambiguities can be resolved. The RH maps presented here are the starting point for mapping additional loci, in particular genes and ESTs that will allow detailed comparative maps between cattle and other species to be constructed. Radiation hybrid cell panels allow high-density genetic maps to be constructed, with the advantage over linkage mapping that markers do not need to be polymorphic. A large quantity of DNA has been prepared from the cells forming the RH panel reported here and is publicly available for mapping large numbers of loci.

Telomere–associated chromosome fragmentation: applications in genome manipulation and analysis

Telomere–associated chromosome fragmentation (TACF) is a new approach for chromosome mapping based on the non–targeted introduction of cloned telomeres into mammalian cells. TACF has been used to generate a panel of somatic cell hybrids with nested terminal deletions of the long arm of the human X chromosome, extending from Xq26 to the centromere. This panel has been characterized using a series of X chromosome loci. Recovery of the end clones by plasmid rescue produces a telomeric marker for each cell line and partial sequencing will allow the generation of sequence tagged sites (STSs). TACF provides a powerful and widely applicable method for genome analysis, a general way of manipulating mammalian chromosomes and a first step towards constructing artificial mammalian chromosomes.

Condensin Regulates the Stiffness of Vertebrate Centromeres

When chromosomes are aligned and bioriented at metaphase, the elastic stretch of centromeric chromatin opposes pulling forces exerted on sister kinetochores by the mitotic spindle. Here we show that condensin ATPase activity is an important regulator of centromere stiffness and function. Condensin depletion decreases the stiffness of centromeric chromatin by 50% when pulling forces are applied to kinetochores. However, condensin is dispensable for the normal level of compaction (rest length) of centromeres, which probably depends on other factors that control higher-order chromatin folding. Kinetochores also do not require condensin for their structure or motility. Loss of stiffness caused by condensin-depletion produces abnormal uncoordinated sister kinetochore movements, leads to an increase in Mad2(+) kinetochores near the metaphase plate and delays anaphase onset.

Co‐localization of centromere activity, proteins and topoisomerase II within a subdomain of the major human X α‐satellite array

Dissection of human centromeres is difficult because of the lack of landmarks within highly repeated DNA. We have systematically manipulated a single human X centromere generating a large series of deletion derivatives, which have been examined at four levels: linear DNA structure; the distribution of constitutive centromere proteins; topoisomerase IIα cleavage activity; and mitotic stability. We have determined that the human X major α‐satellite locus, DXZ1, is asymmetrically organized with an active subdomain anchored ∼150 kb in from the Xp‐edge. We demonstrate a major site of topoisomerase II cleavage within this domain that can shift if juxtaposed with a telomere, suggesting that this enzyme recognizes an epigenetic determinant within the DXZ1 chromatin. The observation that the only part of the DXZ1 locus shared by all deletion derivatives is a highly restricted region of <50 kb, which coincides with the topo isomerase II cleavage site, together with the high levels of cleavage detected, identify topoisomerase II as a major player in centromere biology.

Mitotic chromosomes are compacted laterally by KIF4 and condensin and axially by topoisomerase IIα

During the shaping of mitotic chromosomes, KIF4 and condensin work in parallel to promote lateral chromatid compaction and in opposition to topoisomerase IIα, which shortens the chromatid arms.

Topoisomerase II: untangling its contribution at the centromere.

Topoisomerase II (topo II) is a major component of mitotic chromosomes and its unique decatenating activity has been implicated in many aspects of chromosome dynamics including DNA replication, transcription, recombination, chromosome condensation and segregation. Of these, chromosome segregation is the most seriously affected by loss of topo II expression or activity in living cells, most likely because of residual catenations between sister chromatids. At metaphase, vertebrate chromatids are attached to each other principally through their centromeric regions, and we review here evidence that topo II has a specific role at the centromere. Despite strong evidence for the centromere-specific accumulation of topo II protein and the cytogenetic and molecular mapping of topo II catalytic activity to active centromeres, there is so far relatively little evidence for an overt role in centromere function (as judged by the effects of topo II inactivation on kinetochore assembly, bipolar microtubule attachment and chromosome separation). Nevertheless, recent data linking the post-translational modification of topo II to the regulation of sister centromere cohesion suggest that topo II may indeed contribute to the timely separation of centromeres at anaphase.

Depletion of topoisomerase IIα leads to shortening of the metaphase interkinetochore distance and abnormal persistence of PICH-coated anaphase threads

Topoisomerase II (topo II) is a major component of mitotic chromosomes, and its unique decatenating activity has been implicated in many aspects of chromosome dynamics, of which chromosome segregation is the most seriously affected by loss of topo II activity in living cells. There is considerable evidence that topo II plays a role at the centromere including: the centromere-specific accumulation of topo II protein; cytogenetic/molecular mapping of the catalytic activity of topo II to active centromeres; the influence of sumoylated topo II on sister centromere cohesion; and its involvement in the activation of a Mad2-dependent spindle checkpoint. By using a human cell line with a conditional-lethal mutation in the gene encoding DNA topoisomerase IIα, we find that depletion of topo IIα, while leading to a disorganised metaphase plate, does not have any overt effect on general assembly of kinetochores. Fluorescence in situ hybridisation suggested that centromeres segregate normally, most segregation errors being chromatin bridges involving longer chromosome arms. Strikingly, a linear human X centromere-based minichromosome also displayed a significantly increased rate of missegregation. This sensitivity to depletion of topo IIα might be linked to structural alterations within the centromere domain, as indicated by a significant shortening of the distance across metaphase sister centromeres and the abnormal persistence of PICH-coated connections between segregating chromatids.

Characterization and mapping of the human SOX4 gene

The SOX genes comprise a large family related by homology to the HMG-box region of the testis-determining gene SRY. We have cloned and sequenced the human SOX4 gene. The open reading frame encodes a 474 amino acid protein, which includes an HMG-box. The non-box sequence is particularly rich in serine residues and has several polyglycine and polyalanine stretches. With somatic cell hybrids, human SOX4 has been mapped to Chromosome (Chr) 6p distal to the MHC region. There is no evidence for clustering of other members of the SOX1,-2, and-3 or SOX4 gene families around the SOX4 locus.