Margaret A. Leversha

A systematic, high-resolution linkage of the cytogenetic and physical maps of the human genome.

A systematic, high-resolution linkage of the cytogenetic and physical maps of the human genome

Genetic Analysis of Meiotic Recombination in Humans by Use of Sperm Typing: Reduced Recombination within a Heterozygous Paracentric Inversion of Chromosome 9q32-q34.3

To investigate patterns of genetic recombination within a heterozygous paracentric inversion of chromosome 9 (46XY inv[9] [q32q34.3]), we performed sperm typing using a series of polymorphic microsatellite markers spanning the inversion region. For comparison, two donors with cytogenetically normal chromosomes 9, one of whom was heterozygous for a pericentric chromosome 2 inversion (46XY inv[2] [p11q13]), were also tested. Linkage analysis was performed by use of the multilocus linkage-analysis program SPERM, and also CRI-MAP, which was adapted for sperm-typing data. Analysis of the controls generated a marker order in agreement with previously published data and revealed no significant interchromosomal effects of the inv(2) on recombination on chromosome 9. FISH employing cosmids containing appropriate chromosome 9 markers was used to localize the inversion breakpoint of inv(9). Analysis of inv(9) sperm was performed by use of a set of microsatellite markers that mapped centromeric to, telomeric to, and within the inversion breakpoints. Three distinct patterns of recombination across the region were observed. Proximal to the centromeric breakpoint, recombination was similar to normal levels. Distal to the telomeric breakpoint, there was an increase in recombination found in the inversion patient. Finally, within the inversion, recombination was dramatically reduced, but several apparent double recombinants were found. A putative model explaining these data is proposed.

A mitochondrial elongation factor-like protein is over-expressed in tumours and differentially expressed in normal tissues

The tissue‐specific expression of an antigen (P43) ubiquitously expressed at high levels in a variety of tumours of human and animal origin was investigated using a monoclonal antibody to P43. Whereas low amounts of P43 are expressed in the spleen, skeletal muscle and pancreas, P43 is abundantly produced in the liver and in other tissues such as the kidney, heart and brain which have high levels of oxidative metabolism. Interestingly, a related protein of higher molecular weight is abundantly expressed in the lung and in amounts which were higher than those observed with other tissues. The human cDNA for P43 was isolated from a human liver cDNA library and mapped to chromosome 16 between p11.2 and 12 and also to a position near the centromere on the long arm of chromosome 17. The deduced amino acid sequence of P43 is remarkably similar to that of E. coli EF‐Tu and the mitochondrial EF‐Tu of S. cerevisiae with the structurally and functionally important amino acids of EF‐Tu being completely conserved in P43. A comparison of the distribution of P43 and a mitochondrial protein Hsp 60 among different cellular fractions indicated a likely mitochondrial localisation for P43. Taken together these results suggest that P43 is a human mitochondrial elongation factor.

A rearrangement on Chromosome 5 of an expressed human β-glucuronidase pseudogene

A novel transcript containing homology to exons 5, 9, 10, and 11 of the β-glucuronidase gene has been shown to be derived from Chromosome (Chr) 5. In situ hybridization analysis has shown that this transcript is homologous to four loci on Chr 5 (5p13.3, 5p15.1, 5q13.1, and 5q15), two loci on Chr 6 (6p11.2 and 6p21.3), and one on Chr 22 at 22q11.2. Analysis of cosmid clones from Chr 5 has defined three distinct contigs in which there are tandem genomic repeats of a unit containing sequences homologous to exons 5, 9, and 10 but not 11. Pulsed-field gel electrophoresis (PFGE) analysis has shown that the length of these repeats is highly variable between unrelated individuals, indicating that these regions of Chr 5 are prone to rearrangement. These sequences may be important with respect to stability around the Chr 5 centromere.

Structure and expression analysis of a member of the human glutamate dehydrogenase (GLUD) gene family mapped to chromosome 10p11.2

Glutamate dehydrogenase (GLUD) is a key metabolic enzyme of the mitochondrion, playing an important role in mammalian neuronal transmission. GLUD deficiency has been associated with certain forms of neurodegeneration in the human cerebellum. Genomic DNA blot hybridization analysis and identification of a large number of GLUD-specific genomic clones have suggested that human GLUD is encoded by a multigene family consisting of at least six members. A functional GLUD gene, GLUD1, has been mapped to chromosome 10q22.3-23 and a full-length “processed” GLUD gene, GLUDP1, to chromosome Xq22-23. In the context of studing the structure, the role, and the chromosomal organization of the other family members, we have analysed in detail, a cosmid clone solely reactive with the 3′ region of the GLUD cDNA. Structure and expression analysis of its GLUD-specific region suggests that it represents a truncated “processed” GLUD pseudogene. Fluorescence in situ hybridization using the entire cosmid as a probe, mapped this GLUD gene locus, termed GLUDP5, to chromosome 10p11.2.

Physical mapping of chromosome 3p25-p26 by flourescence in situ hybridisation (FISH)

As part of our effort to isolate and characterise the von Hippel-Lindau (VHL) disease gene, we constructed a physical map of chromosome 3p25-26 by fluorescence in situ hybridisation (FISH) studies on a panel of cytogenetic rearrangements involving this region. Biotinylated cosmid and lambda probes were hybridised to metaphase chromosome spreads and positioned with respect to each cytogenetic breakpoint. These studies unequivocally established the order of five loci linked to the VHL disease gene: cen-(RAF1,312)-D3S732-D3S1250-D3S601-D3S18-pter and determined the position of three other probes within this map. These results ordered RAF1 and D3S732 for the first time, confirmed the localisation of D3S1250 between RAF1 and D3S601 and determined the position of D3S651 with respect to other chromosome 3p25-p26 loci. The establishment of an ordered set of cytogenetic aberrations will enable the rapid assignment of polymorphic and nonpolymorphic cloned sequences within the chromosome region 3p25-p26.

Physical analysis of the tuberous sclerosis region in 9q34.

We report the construction of a physical map based on cloned DNA within the candidate region for the tuberous sclerosis complex (TSC1) gene on chromosome 9q34, between the markers D9S149 and D9S66. The DNA clones form three contigs consisting of 7 YACs, bridged by P1 and cosmid clones, and cover more than 950 kb of 9q34. Despite intensive screening of all available libraries, two gaps remain. A detailed physical map of much of this region was derived, and restriction mapping of the YAC, P1, and cosmid clones reveals novel CpG islands in this region. This set of genomic clones provides a resource for characterizing candidates for the TSC1 gene, guided by the location of CpG islands.

Mapping of two human homologs of a Drosophila heterochromatin protein gene to the X Chromosome

Position-effect variegation (PEV) is, in essence, the regional suppression of euchromatic genes brought into close proximity to heterochromatin by chromosomal rearrangement (Spofford 1976). A renewed interest has recently been shown in genetic modifiers of such variegating position effects (reviewed in Eissenberg 1989). The products of these genes are believed to change the local chromatin structure, leading to an increased (suppressor of variegation) or a decreased (enhancer) transcriptional activity. Cloning and characterization of one modifier gene, HPI, has shown it to be allelic to Suvar(2)5 (Eissenberg et al. 1990) and to be a structural component of heterochromatin (James and Elgin 1986). Regulation of gene expression by changes in chromatin structure has also been invoked as a mechanism for the maintenance of homeogene activity (Gaunt and Singh 1990; Paro 1990). In this system, heritable repression of homeogenes is brought about by a family of trans-regulators known as the Polycomb-group (Pcgroup). Interestingly, cloning and sequencing the groups namesake, Pc, has revealed a region of homology with HP1 (Paro and Hogness 1991). We have used this region, termed chromo box, to clone mammalian cDNAs that show significant homology to Drosophila HPI (Singh et al. 1991). One of these cDNAs, HSM1, is a human clone and shows 51% overall homology, at the amino acid level, with HP1 and 70% homology over the chromo domain. We have suggested that the mammalian HPl-like genes might function as gene repressors, perhaps by serving as components of heterochromatin. The classical example of this type of gene repression, in mammals, is X-Chromosome (Chr) inactivation (Lyon 1991). Our attention has, therefore, been focused on