Biomedical subjects
Publications and source records attributed to P N Goodfellow.
The X/Y homologous gene MIC2 was shown to exchange between the sex chromosomes, thus demonstrating that it is a pseudoautosomal gene in man. MIC2 recombines with the sex-determining gene(s) TDF at a frequency of 2 to 3 percent. It is the most proximal pseudoautosomal locus thus far described and as such is an important marker for use in studies directed towards the isolation of TDF.
Chromosome-mediated gene transfer (CMGT) can be used to generate fragments of human chromosomes and chromosomal maps can be constructed using these fragments. In previous experiments CMGT techniques have been limited to those regions of the genome which encode biochemically selectable markers. We have extended the regions of the human genome which can be subjected to CMGT methods by employing a cell surface antigen as a selectable marker. These experiments have been facilitated by the discovery that co-transformation of chromosomes with a plasmid bearing a biochemically selectable marker followed by selection for the marker pre-selects for cells which have incorporated chromosomal fragments. The plasmid may also integrate into the donor chromosomes and this provides, in some cases, an additional selectable marker in the chromosome fragment of interest. Using these methods we have isolated for the first time cells containing varying portions of the human Y chromosome.
The human malignant melanoma cell line NK14 contains a novel transforming gene which was identified using DNA transfection into NIH3T3 cells (1). This gene has been assigned to chromosome 19p13.2-q13.2 using human-mouse and human-hamster somatic cell hybrids.
We have used mouse and human cDNA probes to map the chromosomal position of the N-CAM gene in the human genome. Southern analysis of DNA isolated from a panel of mouse-human somatic cell hybrids has assigned the N-CAM gene to chromosome 11. This assignment was found with both mouse and human N-CAM cDNAs.
The existence of genes shared by mammalian sex chromosomes has been predicted on both evolutionary and functional grounds. However, the only experimental evidence for such genes in humans is the cell-surface antigen encoded by loci on the X and Y chromosomes (MIC2X and MIC2Y, respectively), which is recognized by the monoclonal antibody 12E7. Using the bacteriophage lambda gt11 expression system in Escherichia coli and immunoscreening techniques, we have isolated a cDNA clone whose primary product is recognized by 12E7. Southern blot analysis using somatic cell hybrids containing only the human X or Y chromosomes shows that the sequences reacting with the cDNA clone are localized to the sex chromosomes. In addition, the clone hybridizes to DNAs isolated from mouse cells that have been transfected with human DNA and selected for 12E7 expression on the fluorescence-activated cell sorter. We conclude that the cDNA clone encodes the 12E7 antigen, which is the primary product of the MIC2 loci. The clone was used to explore sequence homology between MIC2X and MIC2Y; these loci are closely related, if not identical.
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Two globoseries antigens (antigens borne on carbohydrate chains containing globoside), SSEA-3 and SSEA-4, were found on the red cells of the majority of people, but were absent from cells of rare p and Pk individuals which lack globoside. In addition, SSEA-4 was absent from red cells of Luke(-) individuals which nevertheless express the P antigen (globoside) and SSEA-3. The name LKE is proposed for the red cell antigen detected by the Luke serum and by MC813-70, the monoclonal antibody defining SSEA-4. Among the LKE+ individuals, a few showed relatively weak expression of the antigen and were grouped separately as a LKE weak (LKEw) phenotype. Using MC813-70, the frequencies of the 3 phenotypes LKE+, LKEw and LKE- in an English donor population are 0.914, 0.072 and 0.014, respectively.
Seven phage clones containing human sequences were picked at random from a human genomic library cloned in Charon 4A. The clones are devoid of repetitive sequences and can be used to recognize restriction fragment length polymorphisms (Feder et al. 1985). The chromosomal locations of the sequences defined by the seven clones have been determined by Southern blotting and DNA hybridization to DNA from human-mouse somatic cell hybrids. The chromosomal assignment of these sequences should increase their value as genetic markers in family studies.
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A collection of human Y-derived cosmid clones was screened with a plasmid insert containing a member of the human X chromosome alphoid repeat family, DXZ1. Two positive cosmids were isolated and the repeats they contained were investigated by Southern blotting, in situ hybridization and sequence analysis. On hybridization to human genomic DNAs, the expected cross-hybridization characteristic of all alphoid sequences was seen and, in addition, a 5500 base EcoRI fragment was found to be characteristic of a Y-specific alphoid repeat. Dosage experiments demonstrated that there are about 100 copies of this 5500 base EcoRI alphoid fragment on the Y chromosome. Studies utilizing DNA from human-mouse hybrids containing only portions of the Y chromosome and in situ hybridizations to chromosome spreads demonstrated the Y centromeric localization of the 5500 base repeat. Cross-hybridization to autosomes 13, 14 and 15 was also seen; however, these chromosomes lacked detectable copies of the 5500 base EcoRI repeat sequence arrangement. Sequence analysis of portions of the Y repeat and portions of the DXZ1 repeat demonstrated about 70% homology to each other and of each to the human consensus alphoid sequence. The 5500 base EcoRI fragment was not seen in gorilla, orangutan or chimpanzee male DNA.
We have examined the chromosomal location of human T cell-specific genes which are involved in antigen recognition and of a gene which specifically rearranges in T cells. The genes encoding both the variable and constant region segments of the T cell receptor alpha chain are found on chromosome 14 while the delta chain gene of the T cell receptor-associated T3 complex is localised to chromosome 11. Further, the two tandemly arranged T cell-specific rearranging genes, designated gamma, were mapped to chromosome 7, but apparently not closely linked to the previously mapped T cell receptor beta-chain gene. The locations of the three different genes, which undergo rearrangement in T cells, may correlate with the chromosomal breakpoints known to be involved in translocations within abnormal human T cells.
It has been argued on both evolutionary and functional grounds that genes must be shared by the mammalian sex chromosomes. The only direct evidence for such genes is our previous finding that loci on the human X (MIC2X) and Y (MIC2Y) chromosomes encode a species-specific cell surface antigen recognised by the monoclonal antibody 12E7. These loci map to the regions of the sex chromosomes which pair at meiosis, and MIC2X has been shown to escape X-inactivation. We have used immunoprecipitation and Western blot analysis combined with one- and two-dimensional polyacrylamide gel electrophoresis to compare the products of MIC2X and MIC2Y. The human specific molecule recognised by the 12E7 antibody is a membrane-associated polypeptide of mol. wt. 32.5 kd and pI = 5.0. No difference in size or charge has been detected between X and Y encoded forms of this molecule confirming that MIC2Y is a functional homologue of MIC2X. An intracellular polypeptide of mol. wt. 29 kd and pI = 7.0 present in the cytoplasm of both human and mouse cells is also recognised by the 12E7 antibody.
An antigen expressed by most human cells, but not erythrocytes, has been defined by a murine monoclonal antibody, TRA-2-10. This antigen is expressed on the surface of human-mouse somatic cell hybrids, and segregation analysis indicates that it is controlled by a gene located on human chromosome 1. From lysates of most human cells, surface-labelled with 125I, TRA-2-10 immunoprecipitates two polypeptides with molecular weights in the range of about 55 000 to 73 000 depending upon the cell line. Since the TRA-2-10 polypeptides from a fibroblast cell strain and a hepatoma cell line from one individual differ, we conclude that the observed difference in molecular weight has an epigenetic origin.
Monoclonal antibody 30.2A8 was produced by a hybridoma made by fusing cells from rats that had been immunized with rat-human muscle cell hybrids. The 30.2A8 reacts with a differentiation antigen in human skeletal muscle that is synthesized by myoblasts but not myotubes. The gene controlling synthesis of the antigen recognized by 30.2A8 was found to be encoded by human chromosome 15.
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Avian erythroblastosis virus (AEV) induces acute erythroleukemia and sarcomas in vivo and it transforms erythroblasts and fibroblasts in vitro. The virus has two host cell-derived genes, v-erbA and v-erbB. The latter encodes the oncogenic capacity of the virus, whereas v-erbA enhances the erythroblast transforming effects of v-erbB while being unable to induce neoplasms independently. Recently, human cellular homologues of these viral erb genes have been isolated. The chromosomal locations of two of these genes have been determined using EcoRI-digested DNA prepared from human-mouse somatic cell hybrids. The human c-erbA1 gene has been assigned to chromosome 17 and is located between 17p11 and 17q21. The human c-erbB sequence has been assigned to chromosome 7 and is located between 7pter and 7q22. Thus, in the human genome these genes are on two separate chromosomes. No evidence for the involvement of the human c-erb genes in neoplasia has been found.
Clones containing sequences derived from the human Y chromosome have been isolated from cosmid libraries of a human-mouse hybrid cell line. These libraries were constructed in the new expression vectors Homer V and Homer VI. The collection of cosmids isolated is enriched for unique sequence DNA and only a few of the cosmids contain the tandemly repeated sequences which constitute a major portion of the Y chromosome. Three cosmids have been studied in detail. One cosmid shows extensive homology over at least 20 kb with the long arm of the X chromosome; this homology is outside the predicted homology region required for sex chromosome pairing. The other two clones contain unique sequences specific to the Y chromosome and both map to the heterochromatic region of the Y chromosome long arm.
cDNA clones which encode the human and mouse T cell antigen receptor beta chain gene have previously been isolated. We have used a mouse cDNA clone to map the chromosomal position of a human beta chain gene. Southern blot analysis of DNA prepared from somatic cell hybrids has assigned this gene to chromosome 7. The use of a hybrid containing a chromosome 7 translocation has further localised this gene to the region 7q22-qter.