Assignment of markers by using polymerase chain reaction on pools of swine flow-sorted chromosomes

Gene chromosomal assignment can be realized not only by somatic hybrid panels but also by spot-blot hybridization or polymerase chain reaction (PCR) of flow-sorted chromosomes. We propose a swine chromosome assignment strategy by PCR amplification on pooled chromosomal DNA, which allows assignment despite possible chromosomal contamination during sorting. Each pool contains three different chromosomes, each chromosome being present in one or two pools. We present concordant results obtained for eight markers already mapped to different swine chromosomes and we assign the somatostatin gene to chromosome 13, a new marker in the pig genome.     

Localization of the human alpha-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay

We have used 16 human × mouse somatic cell hybrids containing a variable number of human chromosomes to demonstrate that the human α-globin gene is on chromosome 16. Globin gene sequences were detected by annealing purified human α-globin complementary DNA to DNA extracted from hybrid cells. Human and mouse chromosomes were distinguished by Hoechst fluorescent centromeric banding, and the individual human chromosomes were identified in the same spreads by Giemsa trypsin banding. Isozyme markers for 17 different human chromosomes were also tested in the 16 clones which have been characterized. The absence of chromosomal translocation in all hybrid clones strongly positive for the α-globin gene was established by differential staining of mouse and human chromosomes with Giemsa 11 staining. The presence of human chromosomes in hybrid cell clones which were devoid of human α-globin genes served to exclude all human chromosomes except 6, 9, 14 and 16. Among the clones negative for human α-globin sequences, one contained chromosome 2 (JFA 14a 5), three contained chromosome 4 (AHA 16E, AHA 3D and WAV R4D) and two contained chromosome 5 (AHA 16E and JFA14a 13 5) in >10% of metaphase spreads. These data excluded human chromosomes 2, 4 and 5 which had been suggested by other investigators to contain human globin genes. Only chromosome 16 was present in each one of the three hybrid cell clones found to be strongly positive for the human α-globin gene. Two clones (WAIV A and WAV) positive for the human α-globin gene and chromosome 16 were counter-selected in medium which kills cells retaining chromosome 16. In each case, the resulting hybrid populations lacked both human chromosome 16 and the α-globin gene. These studies establish the localization of the human α-globin gene to chromosome 16 and represent the first assignment of a nonexpressed unique gene by direct detection of its DNA sequences in somatic cell hybrids.     

Localization of the human UBC polyubiquitin gene to chromosome band 12q24.3

The localization of specific human ubiquitin genes has not been straightforward because of the conservation of the ubiquitin coding sequence and the number of processed pseudogenes. An congruent to 1.4-kb sequence from the 5'-flanking region of the UBC gene has been shown to be unique to that locus and free from dispersed repeat elements. The cloned 5'-flanking fragment has been used to probe Southern blots of DNA obtained from somatic cell hybrid cell lines. These data indicate that the UBC gene is located on chromosome 12. In situ hybridization with the 5'-flanking probe has refined the assignment to the broad chromosomal subband 12q24.3. These data show that the active ubiquitin genes are not clustered and are located on separate chromosomes. In addition, these studies demonstrate the utility of intron or flanking sequence probes in the specific chromosomal assignment of members of highly conserved gene families.     

Isolation of microcell hybrid clones containing retroviral vector insertions into specific human chromosomes.

We sought an efficient means to introduce specific human chromosomes into stable interspecific hybrid cells for applications in gene mapping and studies of gene regulation. A defective amphotropic retrovirus was used to insert the gene conferring G418 resistance (neo), a dominant selectable marker, into the chromosomes of diploid human fibroblasts, and the marked chromosomes were transferred to mouse recipient cells by microcell fusion. We recovered five microcell hybrid clones containing one or two intact human chromosomes which were identified by karyotype and marker analysis. Integration of the neo gene into a specific human chromosome in four hybrid clones was confirmed by segregation analysis or by in situ hybridization. We recovered four different human chromosomes into which the G418 resistance gene had integrated: human chromosomes 11, 14, 20, and 21. The high efficiency of retroviral vector transformation makes it possible to insert selectable markers into any mammalian chromosomes of interest.     

Confirmed assignments of 15 structural gene loci to chromosomes of four owl monkey karyotypes

Fifteen gene loci for constitutive enzymes previously localized to specific owl monkey chromosomes of karyotypes III, V, and VI are confirmed by their assignments to homologous chromosomes of owl monkey karyotypes I, II, IV, and VII. The syntenic mapping of LDHA and GPI on a large metacentric, II-2, and the separate assignment of these two loci to two acrocentrics, I-9 and I-15, provide genetic evidence supporting the proposed fusion-fission event that characterized the karyotypic difference between owl monkeys inhabiting Colombia and the Panama Canal Zone. Moreover, the proposed hypothesis on chromosome polymorphism among the Colombian owl monkeys with karyotypes II, III, and IV, resulting from a fusion-fission event involving one metacentric and two subtelocentric pairs, is supported by the assignment of LDHB and MDH1 to the large metacentric I-2 and the separate localization of these two gene loci to II-13 and II-14, respectively.     

Visible and "cryptic" segregation of parental chromosomes in embryonic stem hybrid cells

Chromosome segregation of the parental chromosomes was studied in 20 interspecific hybrid clones obtained by fusion of Mus musculus embryonic stem cells with Mus caroli splenocytes. FISH analysis with labeled species specific probes and microsatellite markers was used for identification of the parental chromosomes. Cytogenetic analysis has shown significant intra- and interclonal variability in chromosome numbers and ratios of the parental chromosomes in the hybrid cells: six clones contained all M. caroli chromosomes, nine clones showed moderate segregation of M. caroli chromosomes (from 1 to 7), and five clones showed extensive loss of M. caroli chromosomes (from 12 to complete loss of all M. caroli autosomes). Both methods demonstrated "cryptic" segregation of the somatic partner chromosomes. For instance, five clones with near-tetraploid chromosome sets contained only few M. caroli chromosomes (from 1 to 8). The data obtained suggest that the tetraploid chromosome set per se is not a sufficient criterion for conclusion on the absence of chromosome loss in the hybrid cells. Note that "cryptic" chromosome segregation occurred at a high frequency in the examined hybrid clones. Thus, "cryptic" segregation should be borne in mind for assessing pluripotency and genome reprogramming of embryonic stem hybrid cells.     

The pronatriodilatin gene is located on the distal short arm of human chromosome 1 and on mouse chromosome 4.

Atrial natriuretic factors (ANF) are polypeptides having natriuretic, diuretic, and smooth muscle-relaxing activities that are synthesized from a single larger precursor: pronatriodilatin. Chromosomal assignment of the gene coding for human pronatriodilatin was accomplished by in situ hybridization of a [3H]-labeled pronatriodilatin probe to human chromosome preparations and by Southern blot analysis of somatic cell hybrid DNAs with normal and rearranged chromosomes 1. The human pronatriodilatin gene was mapped to the distal short arm of chromosome 1, in band 1p36. Southern blot analysis of mouse X Chinese hamster somatic cell hybrids was used to assign the mouse pronatriodilatin gene to chromosome 4. This assignment adds another locus to the conserved syntenic group of homologous genes located on the distal half of the short arm of human chromosome 1 and on mouse chromosome 4.     

Assignment of the genes for triose phosphate isomerase to chromosome 6 and tripeptidase-1 to chromosome 10 in Mus musculus by somatic cell hybridization

Evidence is presented for the assignment of the gene for triose phosphate isomerase to Mus musculus chromosome 6 and tripeptidase-1 to chromosome 10 by synteny testing and chromosome assignment in Chinese hamster X mouse somatic cell hybrid clones. Neither TPI nor TRIP-1 were expressed concordantly with any known isozyme markers in 45 hybrid clones (13 primary and 32 secondary). Karyotypic analysis of 21 clones showed that the expression of TPI and chromosome 6 were concordant in all cases as was expressed of TRIP-1 and chromosome 10. Both chromosomes were previously unmarked by isozymes.     

Maize individualized chromosome and derived radiation hybrid lines and their use in functional genomics

The duplicated and rearranged nature of plant genomes frequently complicates identification, chromosomal assignment and eventual manipulation of DNA segments. Separating an individual chromosome from its native complement by adding it to an alien genetic background together with the generation of radiation hybrids from such an addition line can enable or simplify structural and functional analyses of complex duplicated genomes. We have established fertile disomic addition lines for each of the individual maize chromosomes, except chromosome 10, with oat as the host species; DNA is available for chromosome 10 in a haploid oat background. We report on instability and transmission in disomic additions of maize chromosomes 1, 5, and 8; the chromosome 2, 3, 4, 6, 7, and 9 additions appear stable. The photoperiodic response of the two recovered maize chromosome 1 addition lines contrasts to the long-day flowering response of the oat parents and the other addition lines. Only when grown under short days did maize chromosome 1 addition lines set seed, and only one line transmitted the maize chromosome 1 to offspring. Low resolution radiation hybrid maps are presented for maize chromosomes 2 and 9 to illustrate the use of radiation hybrids for rapid physical mapping of large numbers of DNA sequences, such as ESTs. The potential of addition and radiation hybrid lines for mapping duplicated sequences or gene families to chromosome segments is presented and also the use of the lines to test interactions between genes located on different maize chromosomes as observed for ectopic expression of cell fate alterations. Electronic Publication     

Two human genes encoding zinc finger proteins,ZNF12 (KOX 3) and ZNF 26 (KOX 20), map to chromosomes 7p22-p21 and 12q24.33, respectively

Summary Two members of the human zinc finger Krüppel family, ZNF 12 (KOX 3) and ZNF 26 (KOX 20), have been localized by somatic cell hybrid analysis and in situ chromosomal hybridization. The presence of individual human zinc finger genes in mouse-human hybrid DNAs was correlated with the presence of specific human chromosomes or regions of chromosomes in the corresponding cell hybrids. Analysis of such mouse-human hybrid DNAs allowed the assignment of the ZNF 12 (KOX 3) gene to chromosome region 7p. The ZNF 26 (KOX 20) gene segregated with chromosome region 12q13-qter. The zinc finger genes ZNF 12 (KOX 3) and ZNF 26 (KOX 20) were localized by in situ chromosomal hybridization to human chromosome regions 7p22-21 and 12q24.33, respectively. These genes and the previously mapped ZNF 24 (KOX 17) and ZNF 29 (KOX 26) genes, are found near fragile sites.     

The complete karyotype of Aspergilius niger: the use of introduced electrophoretic mobility variation of chromosomes for gene assignment studies

A method is described for unambiguous assignment of cloned genes to Aspergillus niger chromosomes by CHEF gel electrophoresis and Southern analysis. All of the eight linkage groups (LGs), with the exception of LG VII, have previously been assigned to specific chromosomal bands in the electrophoretic karyotype of A. niger. Using a LG VII-specific probe (nicB gene of A. niger) we have shown that LG VII corresponds to a chromosome of about 4.1 Mb. Furthermore, genetic localization of three unassigned genes (glaA, agIA and pepA) in strains in which these genes had been replaced by a selectable marker gene led to a revised karyotype for the chromosomes corresponding to LGs VIII and VI. The revised electrophoretic karyotype reveals only 5 distinct bands. The presence of three pairs of equally sized chromosomes precluded assignment of genes to one specific chromosome in the wild-type strain. However, unambiguous chromosome assignment of cloned genes using CHEF-Southern analysis was demonstrated using a set of A. niger strains with introduced chromosomal size variation. The availability of these tester strains obviates the need to isolate or construct mutant. strains for the purpose of chromosome assignment.     

Sequences homologous to glutamic acid decarboxylase cDNA are present on mouse chromosomes 2 and 10

The chromosomal locations of mouse DNA sequences homologous to a feline cDNA clone encoding glutamic acid decarboxylase (GAD) were determined. Although cats and humans are thought to have only one gene for GAD, GAD cDNA sequences hybridize to two distinct chromosomal loci in the mouse, chromosomes 2 and 10. The chromosomal assignment of sequences homologous to GAD cDNA was determined by Southern hybridization analysis using DNA from mouse-hamster hybrid cells. Mouse genomic sequences homologous to GAD cDNA were isolated and used to determine that GAD is encoded by a locus on mouse chromosome 2 (Gad-1) and that an apparent pseudogene locus is on chromosome 10 (Gad-1ps). An interspecific backcross and recombinant inbred strain sets were used to map these two loci relative to other loci on their respective chromosomes. The Gad-1 locus is part of a conserved homology between mouse chromosome 2 and the long arm of human chromosome 2.     

The complete karyotype of Aspergilius niger: the use of introduced electrophoretic mobility variation of chromosomes for gene assignment studies

A method is described for unambiguous assignment of cloned genes to Aspergillus niger chromosomes by CHEF gel electrophoresis and Southern analysis. All of the eight linkage groups (LGs), with the exception of LG VII, have previously been assigned to specific chromosomal bands in the electrophoretic karyotype of A. niger. Using a LG VII-specific probe (nicB gene of A. niger) we have shown that LG VII corresponds to a chromosome of about 4.1 Mb. Furthermore, genetic localization of three unassigned genes (glaA, agIA and pepA) in strains in which these genes had been replaced by a selectable marker gene led to a revised karyotype for the chromosomes corresponding to LGs VIII and VI. The revised electrophoretic karyotype reveals only 5 distinct bands. The presence of three pairs of equally sized chromosomes precluded assignment of genes to one specific chromosome in the wild-type strain. However, unambiguous chromosome assignment of cloned genes using CHEF-Southern analysis was demonstrated using a set of A. niger strains with introduced chromosomal size variation. The availability of these tester strains obviates the need to isolate or construct mutant. strains for the purpose of chromosome assignment.     

Fingerprinting human chromosomes by polymerase chain reaction-mediated DNA amplification.

We describe here a method for DNA fingerprinting of human chromosomes by Alu-polymerase chain reaction (PCR) amplification of DNA from monochromosomal hybrids, following digestion with restriction endonucleases. DNA digestion with restriction enzymes prior to PCR amplification reduces the total number of amplified fragments. The number and pattern of bands of PCR products observed in an electrophoretic medium are chromosome specific and provide a "fingerprint signature" for individual human chromosomes. Using this approach, we have produced fingerprints for human chromosomes 2, 5, 7, 9, and 12. The applicability of this approach to chromosome identification was assessed by comparing the fingerprints obtained for two different hybrids containing chromosome 7. DNA fragments specific for the long and the short arms of human chromosome 12 have also been identified. In addition, Alu-PCR-generated DNA fragments, specific for different chromosomes, were used to probe Southern blots of a hybrid cell panel to identify human chromosomes present in hybrid cell lines. The chromosomal specificity of these probes permits the identification of intact as well as rearranged chromosomes composed of segments arising from more than one chromosome.     

Somatic cell genetic analysis of the interferon system.

Current advances in the use of somatic cell hybrid systems have enhanced the value of these systems for studying eukaryotic cell functions. We have reviewed the use of somatic cells to investigate the human interferon system. It has been shown that interspecific heterokaryons and hybrid cells can produce interferon(s) of both parental types and may be protected from viral challenge by interferon(s) from either parent. Using mouse-human hybrid cells we have assigned a human gene(s) responsible for regulating interferon to chromosome 21 and genes involved in the production of human interferon to chromosomes 2 and 5. Our data also suggest possible assignment of a locus involved in control of interferon production to chromosome 16. Suggested further uses of the somatic cell system for interferon studies include study of the subunit structure of interferons and the development of hybrid lines that produce human interferon at high levels (interferon/somatic cell hybrids/human gene assignment.     

Genetic homology between man and the chimpanzee: syntenic relationships of genes for galactokinase and thymidine kinase and adenovirus-12-induced gaps using chimpanzee-mouse somatic cell hybrids.

The induction by adenovirus-12 of a site-specific gap and assignment of the chimpanzee genes for thymidine kinase and galactokinase were studied by utilizing chimpanzee-mouse hybrid cells. It has been shown that adenovirus-12 induces a specific gap in the long arm of human chromosome 17 (HS 17); with chimpanzee-mouse hybrid cells the specific gap appears on the short arm of the chimpanzee homolog [PTR 19 (HS 17)] of HS 17. This result supports the proposed relationship of HS 17 to PTR 19 (HS 17) by means of a pericentric inversion. The chimpanzee thymidine kinase and galactokinase genes were assigned to PTR 19 (HS 17), further confirming the homology to HS 17. Other syntenic relationships and gene assignments were consistent with proposed homologies between chimpanzee and human chromosomes.     

Confirmation of the localization of the human recombination activating gene 1 (RAG1) to chromosome 11p13

The human recombination activating gene 1 (RAG1) has previously been mapped to chromosomes 14q and 11p. Here we confirm the chromosome 11 assignment by two independent approaches: autoradiographic and fluorescence in situ hybridization to metaphase spreads and analysis of human-hamster somatic cell hybrid DNA by the polymerase chain reaction (PCR) and Southern blotting. Our results unequivocally localize RAG1 to 11p13.     

Chromosomal and regional localization of the genes for UMPH2, APRT, PEPD, PEPS, PSP, and PGP in mink: comparison with man and mouse

Segregation of mink biochemical markers uridine 5'-monophosphate phosphohydrolase-2 (UMPH2), adenine phosphoribosyltransferase (APRT), phosphoserine phosphatase (PSP), phosphoglycolate phosphatase (PGP), peptidases D (PEPD) and S (PEPS), as well as mink chromosomes, was investigated in a set of mink x mouse hybrid clones. The results obtained allowed us to make the following mink gene assignments: UMPH2, chromosome 8; PEPD and APRT, chromosome 7; PEPS, chromosome 6; and PSP and PGP, chromosome 14. The latter two genes are the first known markers for mink chromosome 14. For regional mapping, UMPH2 was analyzed in mouse cell clones transformed by means of mink metaphase chromosomes (Gradov et al., 1985) and also in mink x mouse hybrid clones carrying fragments of mink chromosome 8 of different sizes. Based on the data obtained, the gene for UMPH2 was assigned to the region 8pter----p26 of mink chromosome 8. The present data is compared with that previously established for man and mouse with reference to the conservation of syntenic gene groups and G-band homoeologies of chromosomes in mammals.     

Mapping of the human casein kinase II catalytic subunit genes: two loci carrying the homologous sequences for the alpha subunit.

The human serine/threonine protein casein kinase II (CK II) contains two distinct catalytic subunits, alpha and alpha 1, which are encoded by different genes. A combination of segregation analysis of rodent-human hybrid cells and chromosomal in situ hybridization have localized the human CK II-alpha DNA sequence to two loci: 11p15.5-p15.4 and 20p13. In contrast, the CK II-alpha' gene has been mapped to chromosome 16 by somatic cell hybrid analysis. Taken together with our previous assignment of the CK II regulatory beta-subunit gene to 6p12-p21, these results indicate that although the products of these genes form a single biological complex, they are encoded on different human chromosomes. Further analysis should determine whether both loci of CK II-alpha are functional, or perhaps one of the two constitutes a pseudogene.     

Chromosomal assignment and trans regulation of the tyrosine aminotransferase structural gene in hepatoma hybrid cells.

The structural gene encoding liver-specific tyrosine aminotransferase (TAT; EC 2.6.1.5) was assigned to mouse chromosome 8 by screening a series of hybrid cell lines for retention of murine Tat-1 gene sequences by genomic Southern blotting. This assignment demonstrated that the Tat-1 structural gene was not syntenic with Tse-1, a chromosome 11-linked locus that negatively regulates TAT expression in trans (A. M. Killary and R. E. K. Fournier, Cell 38:523-534, 1984). We also showed that the fibroblast Tat-1 gene was systematically activated in hepatoma X fibroblast hybrids retaining fibroblast chromosomes 8 in the absence of chromosome 11 but was extinguished in cells retaining both fibroblast chromosomes. Thus, the TAT structural genes of both parental cell types were coordinately regulated in the intertypic hybrids, and the TAT phenotype of the cells was determined by the presence or absence of fibroblast Tse-1.