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1.
Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences. 总被引:21,自引:10,他引:11
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A modular dihydrofolate reductase gene has been introduced into Chinese hamster ovary cells lacking dihydrofolate reductase. Clones capable of growth in the absence of added nucleosides contain one to five copies of the plasmid DNA integrated into the host genome. Upon stepwise selection to increasing methotrexate concentrations, cells are obtained which have amplified the transforming DNA over several hundredfold. A detailed analysis of the chromosomes in three clones indicated the appearance of cytologically distinct chromosomal regions containing the amplified plasmid DNA which differ in surrounding sequence composition, structure, and location. Two of the clones examined have extensive, homogeneously staining regions. The DNA in these homogeneously staining regions replicates in the early part of the S phase. The amplified plasmid DNA is found associated at or near the ends of chromosomes or on dicentric chromosomes. We propose that integration of DNA may disrupt telomeric structures and facilitate the formation of dicentric chromosomes, which may then undergo bridge breakage-fusion cycles. These phenomena are discussed in relation to DNA transfer experiments and modes of gene amplification and chromosome rearrangement. 相似文献
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3.
Summary Several workers have reported that most of the ribosomal DNA genes (rDNA) of the yeast Saccharomyces cerevisiae are located on chromosome I. More recently, data indicating that the yeast rDNA genes are located on chromosome XII has been presented. In this report, we present additional evidence indicating that most of the yeast rDNA genes are not on chromosome I. Starting from a diploid yeast strain, we isolated ten strains which were monosomic (2n-1) for chromosome I. We found that each of these ten strains contained two copies of the rDNA-containing chromosome. In addition, we show that the earlier evidence indicating that the yeast rDNA genes were on chromosome I cannot be explained by a difference in the yeast strains which were used in the different experiments. 相似文献
4.
5S-RNA genes of barley are located on the second chromosome 总被引:1,自引:0,他引:1
A. Kolchinsky V. Kanazin E. Yakovleva A. Gazumyan C. Kole E. Ananiev 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1990,80(3):333-336
Summary The genes coding for 5S RNA in barley were cloned, sequenced, and their cluster was assigned to chromosome 2 using wheat-barley chromosome addition lines. High-resolution gel-electrophoresis of DNA and subsequent hybridization revealed new details of the organization of 5S DNA both in wheat and barley. The in situ hybridization of the cloned 5S gene with triploid endosperm nuclei also suggests that these genes are located in a single locus.On leave from: Department of Plant Breeding and Genetics, College of Agriculture, Orissa University of Agriculture & Technology, Bhubaneswar-751003, India 相似文献
5.
Zolli-Juran M Cechetto JD Hartlen R Daigle DM Brown ED 《Bioorganic & medicinal chemistry letters》2003,13(15):2493-2496
This communication describes the high-throughput screen of a diverse library of 50,000 small molecules against Escherichia coli dihydrofolate reductase to detect inhibitors. Sixty-two compounds were identified as having significant inhibitory activity against the enzyme. Secondary screening of these revealed twelve molecules that were competitive with dihydrofolate, nine of which have not been previously characterized as inhibitors of dihydrofolate reductase. These novel molecules ranged in potency (K(i)) from 26 nM to 11 microM and may represent fresh starting points for new small molecule therapeutics directed against dihydrofolate reductase. 相似文献
6.
Using genetically controlled stocks ofDrosophila melanogaster we have compared the frequency of ectopic pairing in a line showing intense quinacrine fluorescence at two sites (81F and 83E) on chromosome 3 with one showing such fluorescence at only one of these sites (81F). The frequency of ectopic pairing is an order of magnitude greater in cells from the line showing intense fluorescence in both regions than in the line showing it in only one. These data indicate that ectopic pairing is dependent upon properties of discrete chromosome regions as small as individual bands. Since A: T-rich chromatin is known to fluoresce intensely after quinacrine staining, these data further suggest that ectopic pairing is dependent on similarities of the DNA of the discrete chromosome regions involved. 相似文献
7.
Summary We report the construction of recombinant plasmids containing the dihydrofolate reductase structural gene (fol) from several trimethoprim-resistant mutants of Escherichia coli. Strains carrying some of these plasmids produced approximately 6% of their soluble cell protein as dihydrofolate reductase and are therefore excellent sources of the purified enzyme for inhibitor binding or mechanistic studies. The nucleotide sequence of the fol region from each of the plasmids was determined. A plasmid derived from a Ki mutant which produced a dihydrofolate reductase with lowered affinity for trimethoprim contained a mutation in the structural gene that altered the sequence of the polypeptide in a conserved region which is adjacent to the dihydrofolate binding site. Two other independently-isolated mutants which overproduced dihydrofolate reductase had a mutation in the-35 region of the fol promoter. One of them, strain RS35, was also temperature-sensitve for growth in minimal medium. This phenotype was shown to be the result of an additional mutation in a locus unlinked to fol by P1 transduction. The fol regions from two temperature-independent revertants of strain RS35 were sequenced. One of these had a mutation within the dihydrofolate reductase structural gene which altered some properties of the enzyme. This confirmed some previous enzymological data which suggested that some revertants of strain RS35 had mutations in fol (Sheldon 1977). These results suggest that dihydrofolate reductase interacts physically with some other essential gene product in E. coli. 相似文献
8.
Characterization of a DNA repair domain containing the dihydrofolate reductase gene in Chinese hamster ovary cells 总被引:19,自引:0,他引:19
V A Bohr D S Okumoto L Ho P C Hanawalt 《The Journal of biological chemistry》1986,261(35):16666-16672
The formation and removal of UV-induced pyrimidine dimers were measured in restriction fragments near and within the essential dihydrofolate reductase (DHFR) gene in Chinese hamster ovary cells in order to map the genomic fine structure of DNA repair. Dimer frequencies were determined at 0, 8, and 24 h after irradiating the cells with 20 J/m2 UV light (254 nm). Within 8 h, the cells had removed more than 40% of the dimers from sequences near the 5' end of the gene, somewhat fewer from the 3' end, but only 2% from the 3' flanking region and 10% from a region upstream from the gene. The corresponding extent of repair in the genome as a whole is 5-10% in the 8-h period. Isoschizomeric restriction enzyme analysis was used to detect the level of methylation in the fragments in which repair was measured. We found that the only hypomethylated sites in and around the DHFR gene were in the fragment near its 5' end, which displayed maximal DNA repair efficiency. The size of the region of preferential DNA repair at the DHFR locus appears to be in the range of 50-80 kilobases, and this finding is discussed in relation to genomic domains and the structure of mammalian chromatin. 相似文献
9.
Characterization of regions of the Caenorhabditis elegans X chromosome containing vitellogenin genes 总被引:4,自引:0,他引:4
Caenorhabditis elegans contains a family of vitellogenin genes consisting of five closely related genes (vit-1 to vit-5) coding for 186,000 Mr yolk proteins, and one distantly related gene (vit-6) encoding a 200,000 Mr precursor to two smaller yolk proteins. We demonstrate here that, although vit-1 to vit-5 are not clustered (with the exception of vit-3 and vit-4), they are all on the X chromosome. In contrast, vit-6 is autosomal. The genes are strictly regulated during development: they are activated in the intestine of the hermaphrodite worm, following the last larval molt. In order to determine whether the vit genes are contained within chromosomal domains of similarly regulated genes, we have used the chromosomal "walking" technique to isolate 55,000 to 60,000 base-pairs of DNA surrounding each of the X-linked genes and determined the developmental specificity of nearby genes. In the total of 235,400 base-pairs of cloned DNA, seven genes, in addition to the five vit genes, were found. The average gene spacing is approximately 20,000 base-pairs per gene but is highly variable, ranging from less than 2000 to more than 38,000 base-pairs. The seven newly identified genes, called uvt-1 to uvt-7, specify RNAs varying in size from 500 to 2700 bases. With the exception of uvt-4, all of the genes are developmentally regulated; but the patterns of regulation are quite variable, and all are different from the vitellogenin genes. The vit genes, therefore, are not contained within co-regulated chromosomal domains. We also searched for the presence of repetitive DNA, but only four such sequences were found. 相似文献
10.
Background
MicroRNAs (miRNAs) are short non-coding RNAs that regulate differentiation and development in many organisms and play an important role in cancer.Methodology/Principal Findings
Using a public database of mapped retroviral insertion sites from various mouse models of cancer we demonstrate that MLV-derived retroviral inserts are enriched in close proximity to mouse miRNA loci. Clustered inserts from cancer-associated regions (Common Integration Sites, CIS) have a higher association with miRNAs than non-clustered inserts. Ten CIS-associated miRNA loci containing 22 miRNAs are located within 10 kb of known CIS insertions. Only one CIS-associated miRNA locus overlaps a RefSeq protein-coding gene and six loci are located more than 10 kb from any RefSeq gene. CIS-associated miRNAs on average are more conserved in vertebrates than miRNAs associated with non-CIS inserts and their human homologs are also located in regions perturbed in cancer. In addition we show that miRNA genes are enriched around promoter and/or terminator regions of RefSeq genes in both mouse and human.Conclusions/Significance
We provide a list of ten miRNA loci potentially involved in the development of blood cancer or brain tumors. There is independent experimental support from other studies for the involvement of miRNAs from at least three CIS-associated miRNA loci in cancer development. 相似文献11.
It has been previously reported that the abundance and distribution of transposable elements (TEs) in Drosophila heterochromatin are conserved in unrelated stocks although they may greatly differ between families. The biases in genomic distribution of TEs are potentially informative for understanding host–transposon interactions. Here we report that in most stocks, one to four elements of the 1731 retrotransposon family are located on the Y chromosome within regions that appear to be polytenized in larval salivary glands. We discuss the hypothesis that these elements may be beneficial to the host and consider the relevance of our observations to the organization of sequences within the heterochromatin. 相似文献
12.
Amplicons on human chromosome 11q are located in the early/late-switch regions of replication timing 总被引:2,自引:0,他引:2
Amplicons are frequently found in human tumor genomes, but the mechanism of their generation is still poorly understood. We previously measured the replication timing of the genes along the entire length of human chromosomes 11q and 21q and found that many "disease-related" genes are located in timing-transition regions. In this study, further scrutiny of the updated replication-timing map of human chromosome 11q revealed that both amplicons on human chromosomal bands 11q13 and 11q22 are located in the early/late-switch regions of replication timing in two human cell lines (THP-1 and Jurkat). Moreover, examination of synteny in the human and mouse genomes revealed that synteny breakage in both genomes occurred primarily at the early/late-switch regions of replication timing that we had identified. In conclusion, we found that the early/late-switch regions of replication timing coincided with "unstable" regions of the genome. 相似文献
13.
Trypanosoma cruzi proteins which are antigenic during human infections are located in defined regions of the parasite 总被引:2,自引:0,他引:2
T Souto-Padrón M B Reyes S Leguizamon O E Campetella A C Frasch W de Souza 《European journal of cell biology》1989,50(2):272-278
Polyclonal antibodies obtained against antigenic proteins encoded by six recombinant DNA clones of Trypanosoma cruzi were used for the ultrastructural localization of the respective antigens in thin sections of parasites (epimastigote, amastigote and trypomastigote forms of T. cruzi) embedded at low temperature in Lowicryl K4M resin. Antigens of high molecular weight containing tandemly repeated amino acid sequence motifs and recognized by sera from patients with Chagas' disease, were located only in the flagellum, where it contacts the parasite cell body. Other antigens were located on the surface of the parasite while still others were found within the flagellar pocket, as is the case with an antigen released during the acute phase of Chagas' disease. Thus, we conclude that some of the T. cruzi proteins which are antigenic in human infections are located in defined regions of the parasite. Some of the antigens were not expressed to the same extent in the three different developmental stages of the parasite. 相似文献
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E U Poluéktova G V Savchenko V Z Nezametdinova F K Khasanov A A Prozorov 《Genetika》1987,23(10):1839-1846
Integration of functionally active human dihydrofolate reductase (hDHFR) gene into the Bacillus subtilis chromosome was performed. The clones obtained contained 1 to 7 copies of hDHFR gene per chromosome equivalent and were resistant to trimethoprim. In cell lysates of such clones a protein with the molecular mass of hDHFR was detected. The hDHFR gene was stably maintained in all clones having this gene integrated into the bacterial chromosome, when grown under non-selective conditions. 相似文献
16.
The T cell receptor beta chain genes are located on chromosome 6 in mice and chromosome 7 in humans 总被引:48,自引:0,他引:48
N Caccia M Kronenberg D Saxe R Haars G A Bruns J Goverman M Malissen H Willard Y Yoshikai M Simon 《Cell》1984,37(3):1091-1099
Homologous clones that encode the beta chain of the T cell antigen receptor have been isolated recently from both murine and human cDNA libraries. These cDNA clones have been used in connection with interspecies hybrid cell lines to determine that the murine T cell receptor gene is located on chromosome 6 and the human gene on chromosome 7. In situ hybridization confirms these data and further localizes these genes to band B of chromosome 6 in the mouse and bands 7p13-21 in the human genome. The organization of the T cell antigen receptor J beta gene segments and C beta genes appears to be conserved, since very few intraspecies polymorphisms of restriction fragment length have been detected in either mouse or human DNA. 相似文献
17.
De la Concha EG Fernandez-Arquero M Gual L Vigil P Martinez A Urcelay E Ferreira A Garcia-Rodriguez MC Fontan G 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(8):4637-4643
Familial predisposition to IgA deficiency (IgAD) suggests that genetic factors influence susceptibility. Most studies support a polygenic inheritance with a susceptibility locus (designated IGAD1) in the MHC, but its exact location is still controversial. This study aimed to map the predisposing IGAD1 locus (or loci) within the MHC by investigating the pattern of association of the disease with several markers in the region. DNA-based techniques were used to type individual alleles of four polymorphic HLA genes (HLA-DR, -DQA1, -DQB1, and HLA-B), six microsatellites (all located between HLA-DR and HLA-B), and three single nucleotide polymorphisms on the TNF gene. The frequencies of these alleles were compared among ethnically matched populations comprising 182 patients and 343 controls. Additionally, we investigated parents and siblings of 100 of these patients. All four parental haplotypes were established in each family (n = 400), and transmission disequilibrium tests were performed. Surprisingly, our results did not support the hypothesis of a unique susceptibility gene being shared by all MHC susceptibility haplotypes. On HLA-DR1 and -DR7-positive haplotypes IGAD1 mapped to the class II region, whereas on haplotypes carrying HLA-DR3 the susceptibility locus mapped to the telomeric end of the class III region, as reported previously. Our results show how, in complex diseases, individuals may be affected for different genetic reasons and a single linkage signal to a region of a chromosome may actually be the result of disease-predisposing alleles in different linked genes in different pedigrees. 相似文献
18.
Sigma s-dependent promoters in Escherichia coli are located in DNA regions with intrinsic curvature. 总被引:8,自引:2,他引:6
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Expression of a number of genes during stationary phase in Escherichia coli is controlled by the alternative sigma factor sigma s (KatF). Promoters recognized by sigma s do not present a well-defined consensus sequence in their -10 and -35 regions. By polyacrylamide gel electrophoresis of DNA fragments performed at different temperatures, and by computer prediction analyses, we have found that sigma s-regulated promoters are located in regions where DNA shows intrinsic curvatures. This feature does not appear in a stationary-phase-induced promoter which is not controlled by sigma s. We propose that DNA bending may help in recognition and/or binding of sigma s to stationary-phase-induced promoters. 相似文献
19.
Matrix attachment regions are positioned near replication initiation sites, genes, and an interamplicon junction in the amplified dihydrofolate reductase domain of Chinese hamster ovary cells. 总被引:27,自引:11,他引:16
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Genomic DNA in higher eucaryotic cells is organized into a series of loops, each of which may be affixed at its base to the nuclear matrix via a specific matrix attachment region (MAR). In this report, we describe the distribution of MARs within the amplified dihydrofolate reductase (DHFR) domain (amplicon) in the methotrexate-resistant CHO cell line CHOC 400. In one experimental protocol, matrix-attached and loop DNA fractions were prepared from matrix-halo structures by restriction digestion and were analyzed for the distribution of amplicon sequences between the two fractions. A second, in vitro method involved the specific binding to the matrix of cloned DNA fragments from the amplicon. Both methods of analysis detected a MAR in the replication initiation locus that we have previously defined in the DHFR amplicon, as well as in the 5'-flanking region of the DHFR gene. The first of these methods also suggests the presence of a MAR in a region mapping approximately 120 kilobases upstream from the DHFR gene. Each of these MARs was detected regardless of whether the matrix-halo structures were prepared by the high-salt or the lithium 3,5-diiodosalicylate extraction protocols, arguing against their artifactual association with the proteinaceous scaffolding of the nucleus during isolation procedures. However, the in vitro binding assay did not detect the MAR located 120 kilobases upstream from the DHFR gene but did detect specific matrix attachment of a sequence near the junction between amplicons. The results of these experiments suggest that (i) MARs can occur next to different functional elements in the genome, with the result that a DNA loop formed between two MARs can be smaller than a replicon; and (ii) different methods of analysis detect a somewhat different spectrum of matrix-attached DNA fragments. 相似文献
20.
Two variable lymphocyte receptor genes of the inshore hagfish are located far apart on the same chromosome 总被引:2,自引:0,他引:2
Variable lymphocyte receptors (VLR) generate enormous diversity through assembling highly diverse leucine-rich repeat (LRR)
modules and presumably function as antigen receptors in jawless vertebrates. The hagfish, which constitute major extant members
of jawless vertebrates along with lampreys, have two VLR genes designated VLRA and VLRB, whereas only a single VLR gene has been identified in the lamprey. In the present study, we show by fluorescence in situ hybridization (FISH) that
hagfish VLRA and VLRB are located on the same chromosome, but are far apart from each other. Analysis of available inshore hagfish complementary
DNA sequences indicates that VLRA and VLRB do not share a LRR module with an identical nucleotide sequence. Physical separation of VLRA and VLRB is consistent with this observation and indicates that the two VLR genes function as separate units. The FISH protocol developed in this study should be useful for the analysis of the agnathan
genome.
J. K. is a research fellow of the Japan Society for the Promotion of Science. 相似文献