Various means of integration of the expressible human dihydrofolate reductase gene into the Bacillus subtilis genome

Integration of expressible DNA corresponding to the human dihydrofolate reductase (hDHFR) gene into the Bacillus subtilis genome has been achieved in different ways. The clones obtained contained one to seven copies of this gene per genome equivalent and were resistant to trimethoprim. Clones produce a new protein coded by the integrated hDHFR gene. In all clones, the integrated DNA was stably maintained even under nonselective growth conditions.     

Plasmid and chromosomal gene transfer in Bacillus subtilis and Escherichia coli in natural transformation

The transfer of hDHFR gene by natural transformation between Escherichia coli and Bacillus subtilis cells has been studied, as well as intrageneric gene transfer in Bacillus subtilis. The gene was transferred by natural transformation in bacterial cells and included into the chromosome or the plasmid.     

Restoration of radiation resistance in ataxia telangiectasia cells by the introduction of normal human chromosome 11

In order to identify the human chromosome which carries a mutated gene in cells from a patient with the hereditary disorder ataxia telangiectasia belonging to complementation group D (AT-D), we performed chromosome transfer experiments via microcell fusion. A single, pSV2neo-tagged chromosome, either 11 or 12, derived from normal human fibroblasts was introduced into AT-D cells by microcell fusion, and clones which were resistant to the antibiotic G418 were isolated. All 3 hybrid clones containing an additional copy number of chromosome 11 showed a restoration of the resistance of wild-type cells to killing by X-irradiation, whereas all 3 hybrid clones containing an additional copy number of chromosome 12 remained hyper-radiosensitive, like the parental AT cells. The results indicate that a defective gene of AT-D cells is also located on chromosome 11, since a genetic linkage analysis has previously suggested that a defective gene of its complementation group A is located on this chromosome.     

Assignment of the gene for adenosine kinase to chromosome 14 in Mus musculus by somatic cell hybridization.

Evidence is presented for the assignment of the gene for adenosine kinase to Mus musculus chromosome 14 by synteny testing and karyotypic analysis of mouse X Chinese hamster somatic cell hybrid clones. ADOK and two enzymes previously mapped to mouse chromosome 14, NP and ES-10, were expressed concordantly in 29 hybrid clones. Chromosome analysis confirmed this assignment. Syntenic evidence is also presented using several clones of a gene transfer system in which the gene for human HPRT had integrated into modified mouse chromosome 14's.     

Karyotypic abnormality of the X chromosome is rare in mutant HPRT-lymphocyte clones

Lymphocyte clones mutated at the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) locus on the X chromosome were studied by synchronization and G banding to determine the proportion of mutant clones having visible karyotypic change. 47 spontaneously mutant clones, 17 mutant clones induced by X-irradiation and 33 wild-type clones were studied. All clones were karyotypically normal except for 1 clone induced by X-irradiation in which an interstitial deletion of the short arm of the X chromosome had been inserted into the long arm of the same chromosome between q23 and q24; this change may have been coincidental or may have resulted in a position effect mutation. It was concluded that the great majority of mutations were not associated with a visible chromosome abnormality. This conclusion complements molecular studies which suggest that gene changes at the HPRT locus in HPRT- mutants generally extend over segments of DNA too small to be resolved by karyotypic analysis.     

Human chromosome 9 can complement UV sensitivity of xeroderma pigmentosum group A cells

A single human chromosome derived from normal human fibroblasts and tagged with the G418 resistance gene was transferred into SV40-transformed xeroderma pigmentosum group A (XP-A) cells via microcell fusion. When chromosome 1 or 12 was transferred, UV sensitivity of microcell hybrid cells was not changed. By contrast, after transferring chromosome 9, 7 of 11 recipient clones were as UV-resistant as normal human cells. Four other clones were still as UV-sensitive as the parental XP-A cells. Southern hybridization analysis using a polymorphic probe, pEKZ19.3, which is homologous to a sequence of the D9S17 locus on chromosome 9, has confirmed that at least a part of normal human chromosome 9 was transferred into the recipient clones. However, amounts of UV-induced unscheduled DNA synthesis in the UV-resistant clones were only one-third of those in normal human cells. These results indicate that a gene on chromosome 9 can confer complementation of high UV sensitivity of XP-A cells although it is still possible that 2 or more genes might be involved in the defective-repair phenotypes of XP-A.     

Repertoire and Organization of Human T-Cell Receptor α Region Variable Genes

A contig of YAC, PAC, and cosmid clones that spanned the human T-cell receptor α variable (AV) gene region on chromosome 14 was assembled. PCR primers corresponding to the members of 32 different subfamilies were used to map AV genes on the genomic clones. Nucleotide sequencing of PCR products derived from different genomic clones was used to discriminate between related AV gene segments that coamplified. The presence of individual AV gene segments on genomic clones was further confirmed by hybridization both to clones and to human genomic DNA from several unrelated individuals. These results suggest that the T-cell receptor α (TCRA) region in humans contains at least 46 distinct AV gene segments that can be grouped into 32 subfamilies based on nucleotide homology. Several subfamilies appear to contain additional members detectable by hybridization that do not map to chromosome 14.     

A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3(2) chromosome

A Supercos-1 library carrying chromosomal DNA of a plasmid-free derivative of Streptomyces coelicolor A3(2) was organized into an ordered encyclopaedia of overlapping clones by hybridization. The minimum set of overlapping clones representing the entire chromosome (with three short gaps) consists of 319 cosmids. The average insert size is 37.5 kb and the set of clones therefore divides the chromosome into 637 alternating unique and overlapping segments which have an average length of approx. 12.5 kb. More than 170 genes, gene clusters and other genetic markers were mapped to their specific segment by hybridization to the encyclopaedia. Genes could be cloned by direct transformation and complementation of S. coelicolor mutants with cosmids isolated from Escherichia coli , selecting for insertion into the chromosome by homologous recombination. As in other streptomycetes, the ends of the chromosome have long terminal inverted repeats.     

Analysis of the regulation of gene expression during Bacillus subtilis sporulation by manipulation of the copy number of spo-lacZ fusions.

The control of expression of the Bacillus subtilis spoIIA locus was analyzed by titrating gene expression against gene copy number. A plasmid integrated into the B. subtilis chromosome and carrying the spoIIA control region fused to Escherichia coli lacZ was forced to form tandem repeats by the selection of clones that grow on high levels of chloramphenicol, the antibiotic against which the plasmid determines resistance. DNA from the clones was digested with BglII, which did not cut in the reiterated region, and the size of the fragment was determined by orthogonal-field-alternation gel electrophoresis to determine the copy number. Most clones had fairly homogeneous copy numbers. Gene expression was monitored by beta-galactosidase activity. The results indicate that spoIIA was under positive control by a moiety present at about five copies per chromosome. Spore formation was not affected by amplification, so spoIIA-lacZ reiteration did not sequester a molecule required elsewhere for sporulation.     

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.     

The XRCC2 and XRCC3 repair genes are required for chromosome stability in mammalian cells.

The irs1 and irs1SF hamster cell lines are mutated for the XRCC2 and XRCC3 genes, respectively. Both show heightened sensitivity to ionizing radiation and particularly to the DNA cross-linking chemical mitomycin C (MMC). Frequencies of spontaneous chromosomal aberration have previously been reported to be higher in these two cell lines than in parental, wild-type cell lines. Microcell-mediated chromosome transfer was used to introduce complementing or non-complementing human chromosomes into each cell line. irs1 cells received human chromosome 7 (which contains the human XRCC2 gene) or, as a control, human chromosome 4. irs1SF cells received human chromosome 14 (which contains the XRCC3 gene) or human chromosome 7. For each set of hybrid cell lines, clones carrying the complementing human chromosome recovered MMC resistance to near-wild-type levels, while control clones carrying noncomplementing chromosomes remained sensitive to MMC. Fluorescence in situ hybridization with a human-specific probe revealed that the human chromosome in complemented clones remained intact in almost all cells even after extended passage. However, the human chromosome in noncomplemented clones frequently underwent chromosome rearrangements including breaks, deletions, and translocations. Chromosome aberrations accumulated slowly in the noncomplemented clones over subsequent passages, with some particular deletions and unbalanced translocations persistently transmitted throughout individual subclones. Our results indicate that the XRCC2 and XRCC3 genes, which are now considered members of the RAD51 gene family, play essential roles in maintaining chromosome stability during cell division. This may reflect roles in DNA repair, possibly via homologous recombination.     

Characterization of a cluster of human high/ultrahigh sulfur keratin-associated protein genes embedded in the type I keratin gene domain on chromosome 17q12-21

Low stringency screening of a human P1 artificial chromosome library using a human hair keratin-associated protein (hKAP1.1A) gene probe resulted in the isolation of six P1 artificial chromosome clones. End sequencing and EMBO/GenBank(TM) data base analysis showed these clones to be contained in four previously sequenced human bacterial artificial chromosome clones present on chromosome 17q12-21 and arrayed into two large contigs of 290 and 225 kilobase pairs (kb) in size. A fifth, partially sequenced human bacterial artificial chromosome clone data base sequence overlapped and closed both of these contigs. One end of this 600-kb cluster harbored six gene loci for previously described human type I hair keratin genes. The other end of this cluster contained the human type I cytokeratin K20 and K12 gene loci. The center of the cluster, starting 35 kb downstream of the hHa3-I hair keratin gene, contained 37 genes for high/ultrahigh sulfur hair keratin-associated proteins (KAPs), which could be divided into a total of 7 KAP multigene families based on amino acid homology comparisons with previously identified sheep, mouse, and rabbit KAPs. To date, 26 human KAP cDNA clones have been isolated through screening of an arrayed human scalp cDNA library by means of specific 3'-noncoding region polymerase chain reaction probes derived from the identified KAP gene sequences. This screening also yielded four additional cDNA sequences whose genes were not present on this gene cluster but belonged to specific KAP gene families present on this contig. Hair follicle in situ hybridization data for single members of five different KAP multigene families all showed localization of the respective mRNAs to the upper cortex of the hair shaft.     

Physical mapping of human chromosome 17 using fragment-containing microcell hybrids

Hybrid cell lines were generated by microcell-mediated transfer of human chromosome 17 into rat recipient cells. The genotypes of 36 such lines were analyzed using a set of human chromosome 17-derived sequences to probe the structural integrity of the chromosome. Four classes of hybrids were obtained: clones with an apparently intact chromosome 17, clones containing large fragments of the chromosome including both the centromere and the selected marker, clones containing only the selected marker and flanking sequences, and clones containing two 17-derived fragments--the pericentric region plus the region of the selected marker. Data from these hybrids were used in conjunction with published regional localization information to obtain a provisional linear map of the chromosome. Results of this analysis are compared to the gene maps predicted from recent linkage studies and from other somatic cell hybrid experiments.     

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.     

Assignment of the human gene for hexosaminidase B to chromosome 5

Mouse-human somatic cell hybrids between different mouse and human cells were studied for the expression of human hexosaminidases A and B activities. The expression of human hexosaminidase B in the hybrids was found to segregate concordantly with the presence of the human chromosome 5. Mouse-human hybrid clones containing either the human chromosomes 5 and 7 only or the human chromosome 7 only were also included in this study. Expression of human hexosaminidase B activity was detected only in those clones containing human chromosome 5. These results indicate that the gene(s) for human hexosaminidase B is located on chromosome 5. No hexosaminidase A activity was detected in clones which contained either human chromosomes 5 and 7 or chromosome 7.     

兔转基因单细胞克隆株的分离培养及其染色体倍性分析

为检测原代二倍体细胞转基因后单细胞克隆的增殖能力及其染色体倍性稳定性,用脂质体介导的转染方法将质粒DNA pEGFP-C1（带有报告基因GFP和Neo^r）导入体外培养的兔胎儿成纤维细胞中,经G418药物筛选后,分离出73个GFP阳性细胞克隆,最后存活13个（18％）,对其中9个克隆的染色体倍性进行分析,结果只有2个（22％）克隆的染色体倍性正常率在75％以上,分别为80％和75％,其余7个克隆的染色体倍性正常率均在70％以上。这表明,当使用转基因单细胞克隆株作为供核细胞产生克隆动物时,单细胞克隆的增殖代数和染色体倍性的稳定性需要进一步研究提高。     

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.     

Mapping of the versican proteoglycan gene (CSPG2) to the long arm of human chromosome 5 (5q12-5q14).

Versican is a major chondroitin sulfate proteoglycan of vascularized connective tissues whose eponym reflects its functional versatility in macromolecular affinity and interactions. In this report we have localized the versican gene (CSPG2) to the long arm of human chromosome 5 by utilizing a combination of somatic cell hybrids, Southern blotting, polymerase chain reaction, and chromosomal in situ hybridization. The proteoglycan gene segregated concordantly with hybrid cell lines containing the long arm of chromosome 5, comprising the 5q12-q14 band regions. To refine this locus further, we screened a chromosome 5-specific library and isolated several genomic clones encoding a portion of the 5' end of versican. One of these genomic clones was used as a probe for in situ hybridization of human chromosome metaphases. The results corroborated the data obtained using somatic cell hybrids and further refined the assignment of the versican gene to the narrow band region of 5q12-5q14, with the primary site likely to be 5q13.2. The availability of novel genomic clones and the mapping data presented here will make possible the identification of any defect genetically linked to this proteoglycan gene.     

Gene order on the short arm of human chromosome 11: regional assignment of the LDH A gene distal to catalase in two translocations

Summary Leukemic cells with reciprocal translocations involving 11p13 and 14q13 were obtained from two patients with T-cell acute lymphoblastic leukemia and fused with mouse Ltk^- cells. DNA from independent hybrid clones was screened by Southern blot and hybridization to molecular probes for the human catalase and Ha-ras-1 genes. Several clones showed segregation of these two genes, indicating the presence of either the der 11 or der 14 human chromosomes. When DNA from these hybrid clones was examined for the presence of the human genes for calcitonin and γ-globin, both genes were found to segregate with the Ha-ras-1 gene and the der14 chromosome indicating that they lie distal to catalase. When the hybrid clones were examined for the presence of human lactate dehydrogenase A (LDH A) activity, only those clones containing the der14 chromosome expressed activity indicating that the LDH A gene is also distal to catalase on the short arm of chromosome 11.