Assignment of the genes for the alpha and beta subunits of thyrotropin to different mouse chromosomes.

A series of mouse-hamster somatic cell hybrids, containing reduced numbers of mouse chromosomes and a complete set of hamster chromosomes, was used to determine the chromosomal locations of the genes for the alpha and beta subunits of mouse thyrotropin. Cloned cDNA probes for each subunit, in conjunction with Southern blot analysis of DNA treated with the restriction enzyme BamHI, allowed for assignment of the alpha-subunit gene to mouse chromosome 4 and of the beta-subunit gene to chromosome 3. Mouse alpha-subunit gene sequences always segregated with chromosome 4 (concordant in 14 hybrids) and the enzyme markers phosphoglucomutase 2 and 6-phosphogluconate dehydrogenase. Mouse beta-subunit gene sequences always segregated with chromosome 3 (concordant in 15 hybrids). Thus, the genes for at least one of the glycoprotein hormones, thyrotropin, are on different chromosomes.     

Assignment of the gene for the beta subunit of thyroid-stimulating hormone to the short arm of human chromosome 1.

The chromosomal locations of the genes for the beta subunit of human thyroid-stimulating hormone (TSH) and the glycoprotein hormone alpha subunit have been determined by restriction enzyme analysis of DNA extracted from rodent-human somatic cell hybrids. Human chorionic gonadotropin (CG) alpha-subunit cDNA and a cloned 0.9-kilobase (kb) fragment of the human TSH beta-subunit gene were used as hybridization probes in the analysis of Southern blots of DNA extracted from rodent-human hybrid clones. Analysis of the segregation of 5- and 10-kb EcoRI fragments hybridizing to CG alpha-subunit cDNA confirmed the previous assignment of this gene to chromosome 6. Analysis of the patterns of segregation of a 2.3-kb EcoRI fragment containing human TSH beta-subunit sequences permitted the assignment of the TSH beta-subunit gene to human chromosome 1. The subregional assignment of TSH beta subunit to chromosome 1p22 was made possible by the additional analysis of a set of hybrids containing partially overlapping segments of this chromosome. Human TSH beta subunit is not syntenic with genes encoding the beta subunits of CG, luteinizing hormone, or follicle-stimulating hormone and is assigned to a conserved linkage group that also contains the structural genes for the beta subunit of nerve growth factor (NGFB) and the proto-oncogene N-ras (NRAS).     

Human histone genes map to multiple chromosomes

Histone genes were mapped to at least three human chromosomes by Southern blot analysis of DNAs from a series of mouse-human somatic cell hybrids (using 32P-labeled cloned human histone DNA as probes). Chromosome assignment was confirmed by in situ hybridization of radiolabeled histone gene probes (3H-labeled) to metaphase chromosomes. One human histone gene cluster (lambda HHG41) containing an H3 and H4 gene resides only on chromosome 1, whereas other clusters containing core (H3, H4, H2A, and H2B) alone (lambda HHG17) or core together with H1 histone genes (lambda HHG415) have been assigned to chromosomes 1, 6, and 12. These results suggest that the multigene family of histone coding sequences that reside in a series of clusters may be derived from a single cluster containing one each of the genes for the five principal classes of histone proteins. During the course of evolution, a set of events, probably involving reduplication, sequence modification, and recombination, resulted in the present pattern of human histone gene distribution among several chromosomes.     

Chromosomal assignment of the human homologues of feline sarcoma virus and avian myeloblastosis virus onc genes.

Retroviral transforming genes, v-onc genes, are derived from normal cellular sequences that are called cellular onc (c-onc) genes. DNA from mouse-human somatic cell hybrids that have selectively lost human chromosomes was used in Southern blots to map the chromosomal location of two human onc genes. Cloned human homologues of retroviral onc genes were used as probes. Because the human c-fes gene, which is homologous to feline sarcoma virus, segregates concordantly with human chromosome 15, and the human c-myb gene, which is homologous to avian myeloblastosis virus onc genes, segregates concordantly with human chromosome 6, we have assigned the c-fes and the c-myb genes to human chromosomes 15 and 6, respectively. Nonrandom chromosomal defects involving these human chromosomes have been observed in neoplasms. These studies should be valuable in determining whether specific rearrangements involving these chromosomes result in the abnormal expression of these onc genes in human malignancies.     

Regional assignment of the structural gene for human alpha-L-iduronidase.

The structural gene encoding human alpha-L-iduronidase has been assigned to chromosome 22 by using immunologic, electrophoretic, and somatic cell hybridization techniques. Polyclonal rabbit antibodies raised against purified human low-uptake alpha-L-iduronidase were used to discriminate the human and murine isozymes by a sensitive immuno-precipitation assay. The human chromosome constitution of each clone was determined by cytogenetic and enzyme marker electrophoretic techniques. In 65 human (fibroblast)-mouse (RAG) somatic cell hybrids (from four independent fusions), the expression of human alpha-L-iduronidase was 100% concordant with the presence of human chromosome 22; the assignment was confirmed by the demonstration of the human enzyme in tertiary somatic cell hybrids containing only chromosome 22. Further verification of the gene assignment was made by detection of the human enzyme in tertiary chromosome 22 positive hybrids by Ouchterlony immunodiffusion and rocket immunoelectrophoretic experiments with polyclonal anti-human alpha-L-iduronidase antibodies that were monospecific for the human enzyme. Expression of human enzymatic activity in chromosome 22 positive hybrid lines was markedly reduced; for example, a tertiary hybrid (R-G21-J-15), which contained an average of 1.7 chromosome 22s per cell, only had about 15% of the activity detected in normal diploid fibroblasts. Immunologic studies suggested that the reduced expression was due to abnormal post-translational processing or aggregation (or both) of the human and murine isozymes in these hybrids. Regional assignment of the human structural gene to 22pter----q11 was accomplished by gene dosage studies using diploid human fibroblast lines that were partially monosomic or trisomic for chromosome 22.     

Precise localization of human beta-globin gene complex on chromosome 11.

Cloned DNA probes were used in combination with a panel of five hybrid cell clones containing a series of different terminal deletions in human chromosome 11 to map precisely the human hemoglobin beta and delta chain structural genes contained on this chromosome. The region of deletion in each clone of the panel has been defined by biochemical, immunologic, and cytogenetic markers. DNA from clones containing successively larger terminal deletions was tested with appropriate DNA probes to determine the point on the chromosome at which DNA for these two closely linked hemoglobin genes is deleted. These genes, and by inference the closely linked G gamma and A gamma globin genes as well, have been assigned to the intraband region 11p1205 leads to 11p1208 on the short arm of chromosome 11, an interval containing approximately 4500 kilobases of DNA. The approach appears to have potential for even greater resolution and reasonably wide applicability for gene mapping.     

Chromosomal localization of human β globin gene on human chromosome 11 in somatic cell hybrids

We have successfully used a DNA.cDNA molecular hybridization assay to directly determine the presence or absence of human beta globin gene sequences in 20 human-mouse somatic cell hybrids, each of which contained a different subset of human chromosomes. The assay is specific for the individual human globin genes and will detect the presence of a globin gene if the relevant chromosome is present in only 10% of the cells of a hybrid population. The content of human chromosomes in each hybrid clone was characterized by Giemsa 11 staining, Giemsa trypsin-Hoechst 33258 staining, and by the use of 22 independent isozyme markers for 17 different human chromosomes. All human chromosomes were present in one or more cell lines devoid of the human beta globin gene except for 6, 8, 9, 11, and 13. Among these latter chromosomes, only chromosome 11 was present in the six hybrid clones that contained the human beta globin gene. In fact, chromosome 11 was the only human chromosome that was present in all of the six hybrid clones found to be positive for the human beta globin gene. Two sister clones, 157-BNPT-1 and 157-BNPT-4, had similar subsets of human chromosomes except that 11 was present only in 157-BNPT-4. 157-BNPT-4 contained the human beta globin gene while 157-BNPT-1 did not. DNA from three hybrid lines was also annealed to purified human gamma globin cDNA; two lines positive for human beta globin gene sequences also contained human gamma globin gene sequences while one line was negative for both beta and gamma gene sequences. On the basis of these results, the human beta and gamma globin genes have been assigned to human chromosome 11.     

Sequences related to HLA-DR alpha chain on human chromosome 6: restriction enzyme polymorphism detected with DC alpha chain probes.

Three sets of cosmid clones--containing the HLA-DR alpha chain gene and two additional related genes--were isolated from human genomic DNA libraries by using a cDNA probe for the HLA-DR alpha chain. Southern blot analysis using DNA from somatic cell hybrids indicated that all of the clones mapped to chromosome 6. Partial sequence analysis showed that the two additional related genes were highly homologous to each other, and to the HLA-DR alpha chain, in parts of the exon that encoded the alpha 2 domain but were more divergent in intron sequences. One of the genes corresponds to the HLA-DR-related DC series. DNA probes made from this gene revealed marked restriction enzyme polymorphism when hybridized to genomic DNA from HLA-DR typed homozygous cell lines. The patterns obtained from a number of homozygous and heterozygous cell lines correlated with the HLA-DR crossreactive serotypes and also indicated that there is a further sequence in the haploid human genome that is closely homologous with the DC alpha chain sequence. One family was studied and showed the expected HLA-DR-associated inheritance of restriction enzyme patterns. No polymorphism has yet been demonstrated in restriction enzyme fragments that include the other cloned sequence, which may correspond to the SB alpha chain gene or to a novel HLA-DR-related gene. These experiments indicate that there are at least three sequences in the human genome related, but not identical, to the HLA-DR alpha chain gene.     

Isolation of a human repetitive sequence and its application to regional chromosome mapping.

Recombinant lambda phage Charon 4A with repetitive human DNA inserts have been constructed by using cellular DNA from a human-Chinese hamster ovary cell hybrid retaining the complete hamster genome and a single human chromosome 12. One recombinant phage, 12-11, contains several repetitive sequences, each with a different repetition pattern in the human genome. A 2.2-kilobase (kb) EcoRI fragment of this phage was subcloned in pBR325. This sequence has fewer than 5,000 copies in the human genome and does not cross-hybridize with Chinese hamster DNA. When the labeled 2.2-kb probe was hybridized to human chromosome 12 DNA digested with EcoRI, there was an intense band at the 2.2-kb position and a series of other discrete bands. The band pattern at positions other than 2.2 kb appears to be distinct for each human chromosome. The 2.2-kb fragment is composed of at least three subregions. The ends of the fragment are repeated more frequently in the genome than is the middle portion. Hybridization of chromosome 12 DNA with probes made to these subregions yielded simpler band patterns. By using a series of cell hybrids containing various deletions of human chromosome 12, five sequences related to the 2.2-kb fragment have been assigned regionally to a specific portion of the short arm of chromosome 12. These results demonstrate that certain repetitive sequences in the human genome can be used as genetic markers and may permit detailed regional mapping of human chromosomes.     

The related genes encoding growth hormone and prolactin have been dispersed to chromosomes 10 and 17 in the rat

We have assigned the rat GH gene to chromosome 10 and the rat PRL gene to chromosome 17. DNA from a series of mouse BWTG3 x rat hepatocyte somatic cell hybrids, each of which has retained a unique complement of rat chromosomes, was analyzed for the presence of rat GH and PRL genomic fragments by Southern blotting. Radiolabeled complementary DNAs (cDNAs) encoding rat GH and rat PRL were used as molecular probes. Based upon these assignments, we conclude that the evolutionarily related GH and PRL genes have been dispersed to different chromosomes in rat as in man.     

Chromosomal locations of the human and mouse genes for precursors of epidermal growth factor and the beta subunit of nerve growth factor.

DNA probes for pre-pro-epidermal growth factor (EGF) and the precursor of the beta subunit of nerve growth factor (NGF) were used to chromosomally map human and mouse EGF and NGF genes in panels of human-mouse and mouse-Chinese hamster somatic cell hybrids. The EGF and NGF genes were mapped to human chromosomes 4 and 1, respectively, by using human-mouse cell hybrids. A combination of regional mapping using a chromosome 1 translocation and comparative gene mapping suggests that the human NGF gene is in the p21-p22.1 region of chromosome 1. In mouse-Chinese hamster cell hybrids, both genes were assigned to mouse chromosome 3. A knowledge of the chromosomal assignment of these genes should help in our understanding of their regulation and role in development and disease.     

Assignment of the gene for beta 2-microglobulin (B2m) to mouse chromosome 2.

We have assigned the gene (B2m) coding for murine beta 2-microglobulin (B2M) to mouse chromosome 2 by using a novel panel of Chinese hamster-mouse somatic cell hybrid clones. Because of 35 independent primary hybrids used in this study were derived from two types of feral mice, each with a different combination of Robertsonian translocation chromosomes, as well as from mice with a normal complement of acrocentric chromosomes, analysis of 16 selected mouse enzyme markers provided data on the segregation of all 20 mouse chromosomes in these hybrids. Mouse B2M was identified in cell hybrids by immunoprecipitation with a species-specific anti-mouse B2M antiserum followed by two-dimensional polyacrylamide gel electrophoresis of the immunoprecipitated polypeptides. Enzyme analysis of the segregant clones excluded all chromosomes for B2m assignment except mouse chromosome 2, and karyotype analysis of nine informative hybrid clones confirmed the assignment of B2m to this chromosome. These results demonstrate that, in the mouse, as in man, B2m is not linked to the major histocompatibility or immunoglobulin loci.     

Chromosomal assignments of the genes coding for human types II, III, and IV collagen: a dispersed gene family.

The human type II collagen gene, COL2A1, has been assigned to chromosome 12, the type III gene, COL3A1, to chromosome 2, and one of the type IV genes, COL4A1, to chromosome 13. These assignments were made by using cloned genes as probes on Southern blots of DNA from a panel of mouse/human somatic cell hybrids. The two genes of type I collagen, COL1A1 and COL2A1, have been mapped previously to chromosomes 17 and 7, respectively. This family of conserved genes seems therefore to be dispersed throughout the genome.     

Isolation of a yeast artificial chromosome spanning the 8;21 translocation breakpoint t(8;21)(q22;q22.3) in acute myelogenous leukemia.

The 8;21 translocation is one of the most common specific rearrangements in acute myelogenous leukemia. We have identified markers (D21S65 and a Not I boundary clone, Not-42, referred to as probe B) flanking the chromosome 21 translocation breakpoint (21q22.3) that demonstrate physical linkage in normal genomic DNA, by using at least three restriction endonucleases (Not I, Sac II, and BssHII), and that are located not more than 250-280 kilobases apart. Pulsed-field gel analysis of DNA from somatic cell hybrids containing the 8;21 translocation chromosomes demonstrates rearrangement of these markers. A 470-kilobase yeast artificial chromosome, YAC-Not-42, has been isolated that contains both probes. Mapping of lambda subclones constructed from YAC-Not-42 suggests that greater than 95% (25/26 probes tested) of the yeast artificial chromosome DNA is located on the proximal (D21S65) side of the breakpoint. In situ hybridization studies using metaphase chromosomes from five acute myelogenous leukemia patients with the 8;21 translocation confirmed these results and demonstrated the translocation of probe B to the derivative chromosome 8. A chromosome walk of approximately 39 kilobases from probe B has allowed identification of the breakpoint in DNA from a somatic cell hybrid containing the derivative chromosome 8. Since probe B contains conserved DNA sequences and is in close proximity to the translocation breakpoint, it may represent a portion of the involved gene on chromosome 21.     

Alu polymerase chain reaction: a method for rapid isolation of human-specific sequences from complex DNA sources.

Current efforts to map the human genome are focused on individual chromosomes or smaller regions and frequently rely on the use of somatic cell hybrids. We report the application of the polymerase chain reaction to direct amplification of human DNA from hybrid cells containing regions of the human genome in rodent cell backgrounds using primers directed to the human Alu repeat element. We demonstrate Alu-directed amplification of a fragment of the human HPRT gene from both hybrid cell and cloned DNA and identify through sequence analysis the Alu repeats involved in this amplification. We also demonstrate the application of this technique to identify the chromosomal locations of large fragments of the human X chromosome cloned in a yeast artificial chromosome and the general applicability of the method to the preparation of DNA probes from cloned human sequences. The technique allows rapid gene mapping and provides a simple method for the isolation and analysis of specific chromosomal regions.     

Human dihydrofolate reductase gene is located in chromosome 5 and is unlinked to the related pseudogenes.

The chromosomal location of the human dihydrofolate reductase (DHFR; EC 1.5.1.3) gene that is amplified in a methotrexate-resistant human cell line has been investigated by screening a large number of human-mouse cell hybrids containing overlapping subsets of human chromosomes. A correlation of genomic blotting data with the chromosome constitution of the individual cell hybrids has allowed the assignment of the human DHFR gene to chromosome 5. This chromosome assignment has been confirmed by the observation of a concomitant loss of the human DHFR gene and of sensitivity to diphtheria toxin, a marker associated with chromosome 5, in two human-mouse cell hybrids selected for resistance to the toxin. Six EcoRI fragments of human DNA containing DHFR pseudogenes or other DHFR-related sequences have been assigned to chromosomes other than chromosome 5.     

Isolation and analysis of the 21q+ chromosome in the acute myelogenous leukemia 8;21 translocation: evidence that c-mos is not translocated.

Acute myelogenous leukemia (AML), subgroup M2, is associated with a nonrandom chromosomal translocation, t(8;21)(q22,q22). The oncogene c-mos also has been localized to the q22 band on chromosome 8. There is also evidence that genes on chromosome 21 may be important in the development of leukemia. To determine whether the c-mos oncogene has been translocated in AML-M2 with this translocation and to isolate DNA sequences and genes from these two chromosomes that may be important in malignancy, we constructed somatic cell hybrids between a Chinese hamster ovary cell (CHO) mutant defective in glycine metabolism and myeloblasts with an 8;21 translocation from a patient with AML. We isolated the 21q+ chromosome of this translocation in a somatic cell hybrid and showed that the c-mos oncogene had not been translocated to chromosome 21, ruling out the possibility that translocation of c-mos to chromosome 21 is necessary for development of AML-M2. In addition, there was no detectable rearrangement of the c-mos locus within a 12.4-kilobase region surrounding the gene, indicating that rearrangement of the coding region of the gene itself or alteration of proximal 5' or 3' flanking sequences is not involved. We used this hybrid to determine whether specific DNA sequences and biochemical markers from chromosomes 8 and 21 had been translocated in this case.     

Assignment of the gene for methylthioadenosine phosphorylase to human chromosome 9 by mouse-human somatic cell hybridization.

The purine and polyamine metabolic enzyme methylthioadenosine (MeSAdo) phosphorylase is abundant in normal cells and tissues but is lacking from many human and murine malignant cell lines and from cells of some human leukemias in vivo. To explore the genetic control of MeSAdo phosphorylase expression, we measured levels of the enzyme in somatic cell hybrids prepared by fusing MeSAdo phosphorylase-deficient mouse L cell lines with human fibroblasts. In the hybrid clones, MeSAdo phosphorylase activity segregated concordantly with adenylate kinase 1, a marker for human chromosome 9, but not with enzyme markers for any other human chromosome. In hybrid clones derived from human fibroblasts with a reciprocal translocation between chromosomes 9 and 17, MeSAdo phosphorylase activity was confined to cells containing the 9pter----9q12 region. In every case, the enzyme-positive hybrid clones displayed bands of MeSAdo phosphorylase activity with isoelectric points characteristic of both the human and murine enzymes. These results indicate that the structural gene for human MeSAdo phosphorylase, designated MTAP, can be assigned to the 9pter----9q12 region of human chromosome 9. Furthermore, these studies with interspecies somatic cell hybrids show that the MeSAdo phosphorylase-deficient state is recessive in mouse L cell lines.     

Sequence variations in the 5' flanking and IVS-II regions of the G gamma- and A gamma-globin genes of beta S chromosomes with five different haplotypes

We have amplified and sequenced the 5' flanking and the second intervening sequence (IVS-II) regions of both the G gamma- and A gamma-globin genes of the beta S chromosomes from sickle cell anemia (SS) patients with homozygosities for five different haplotypes. The sequencing data, compared with previously published sequences for the normal chromosomes A and B, show many similarities to chromosome B for haplotypes 19, 20, and 17, while haplotypes 3 and 31 are remarkably similar to chromosome A and also similar to each other. Several unique mutations were found in the 5' flanking regions (G gamma and A gamma) of haplotypes 19 and 20 and in the IVS-II segments of the same genes of haplotypes 19, 20, and 17; the IVS-II of haplotypes 3 and 31 were identical to those of chromosome A. Dot-blot analyses of amplified DNA from additional SS patients with specific probes have confirmed that these mutations are unique for each haplotype. The two general patterns that have been observed among the five haplotypes have most probably arisen by gene conversion events between the A and B type chromosomes in the African population. These patterns correlate with high and low fetal hemoglobin expression, and it is speculated that these and other yet unknown gene conversions may contribute to the variations in hemoglobin F and G gamma levels observed among SS patients. In vitro expression experiments involving the approximately 1.3-kb 5' flanking regions of the G gamma- and A gamma-globin genes of the beta S chromosomes with the five different haplotypes failed to detect differences between the levels of expression, suggesting that the sequence variations observed between these segments of DNA are not the primary cause of the differences in hemoglobin F levels among the SS patients.     

Site-specific integration by adeno-associated virus.

Cellular sequences flanking integrated copies of the adeno-associated virus (AAV) genome were isolated from a latently infected clonal human cell line and used to probe genomic blots derived from an additional 21 independently derived clones of human cells latently infected with AAV. In genomic blots of uninfected human cell lines and of primary human tissue, each flanking-sequence probe hybridized to unique bands, but in 15 of the 22 latently infected clones the flanking sequences hybridized not only to the original fragments but also to a total of 36 additional species. AAV probes also hybridized to 22 of these new bands, representing 11 of the 15 positive clones, but never to the fragment characteristic of uninfected cell DNA. From these data we conclude that the AAV genome preferentially integrates into a specific region of the cellular genome. We have determined that the integration site is unique to chromosome 19 by somatic cell hybrid mapping, and this sequence has been isolated from uninfected human DNA.