The gene for ornithine decarboxylase is co-amplified in hydroxyurea-resistant hamster cells

We have constructed a cDNA library from the highly hydroxyurea-resistant hamster cell line 600H in which the activity of ribonucleotide reductase is elevated more than 80-fold. Using the technique of differential hybridization, we have isolated a number of cDNA clones from this library which are homologous to genomic DNA sequences amplified in the 600H cell line compared to the V79 parental line. One of these cDNA clones by sequence analysis was found to code for ornithine decarboxylase. This was confirmed by in vitro translation of poly(A+) RNA isolated by hybridization-selection followed by immunoprecipitation with antiserum specific for mouse ornithine decarboxylase. Genomic sequences homologous to the cDNA clone were shown to be sequentially amplified 6-20-fold in hamster cell lines selected stepwise for resistance to increasing concentrations of hydroxyurea. Genomic sequences homologous to a cDNA for the M2 subunit of ribonucleotide reductase were also amplified in these cell lines, and the degree of M2 sequence amplification corresponded to the degree of amplification of ornithine decarboxylase sequences, suggesting that the two genes had been co-amplified during the selection of the hydroxyurea-resistant phenotype.     

Ribonucleotide reductase M2 subunit sequences mapped to four different chromosomal sites in humans and mice: functional locus identified by its amplification in hydroxyurea-resistant cell lines

The sites of sequences homologous to a murine cDNA for ribonucleotide reductase (RR) subunit M2 were determined on human and murine chromosomes by Southern blot analysis of interspecies somatic cell hybrid lines and by in situ hybridization. In the human genome, four chromosomal sites carrying RRM2-related sequences were identified at 1p31----p33, 1q21----q23, 2p24----p25, and Xp11----p21. In the mouse, M2 sequences were found on chromosomes 4, 7, 12, and 13 by somatic cell hybrid studies. By Southern analysis of human hydroxyurea-resistant cells that overproduce M2 because of gene amplification, we have identified the amplified restriction fragments as those that map to chromosome 2. To further confirm the site of the functional RRM2 locus, two other cDNA clones, p5-8 and S7 (coding for ornithine decarboxylase; ODC), which are coamplified with RRM2 sequences in human and rodent hydroxyurea-resistant cell lines, were mapped by Southern and in situ hybridization. Their chromosomal map positions coincided with the region of human chromosome 2 (p24----p25) that also contains one of the four RRM2-like sequences. Since this RRM2 sequence and p5-8 and ODC are most likely part of the same amplification unit, the RRM2 structural gene can be assigned to human chromosome 2p24----p25. This region is homologous to a region of mouse chromosome 12 that also carries one of numerous ODC-like sequences. In an RRM2-overproducing mouse cell line, we found amplification of the chromosome 12-specific restriction fragments. Thus, we conclude that mouse chromosome 12 carries the functional locus for RRM2.     

Cloning and characteristics of DNA sequences amplified in Djungarian hamster cells with multidrug resistance

A number of DNA clones containing the amplified DNA sequences were isolated from the genomic library of multidrug-resistant (MDR) Djungarian hamster cells using the DNAC0t 10-250 hybridization probe. Five independent nonoverlapping clones were obtained that covered more than 100 kb of the amplified genomic region. These clones were used as hybridization probes in blot-hybridization with DNA from 7 independently derived MDR Djungarian hamster cell lines selected for the resistance to colchicine or actinomycin D. Some clones contained the DNA sequences amplified in all of the cell lines tested while the others contained the cell line specific amplified sequences. Hybridization in situ was used to localize the amplified DNA in metaphase chromosomes of a MDR cell line that contained about 140 copies of these sequences. The approximate size of an amplicon calculated on the basis of the obtained data is about 1-2 X 10(3) kb.     

Differential amplification and disproportionate expression of five genes in three multidrug-resistant Chinese hamster lung cell lines.

At least five linked genes are amplified in the multidrug-resistant Chinese hamster ovary cell line CHRC5, selected with colchicine (A. M. Van der Bliek, T. Van der Velde-Koerts, V. Ling, and P. Borst, Mol. Cell. Biol. 6:1671-1678, 1986). We report here that only a subset of these, encoding the 170-kilodalton P-glycoprotein, are consistently amplified in three different multidrug-resistant Chinese hamster lung cell lines, selected with vincristine, daunorubicin, or actinomycin D. Within each cell line, genomic sequences homologous to the P-glycoprotein cDNA probe were amplified to different levels. The pattern of differential amplification was consistent with the presence of at least two and possibly three P-glycoprotein genes. In the actinomycin D-selected cell line, these genes were disproportionately overexpressed relative to the associated levels of amplification. These results underline a central role for P-glycoprotein in multidrug resistance. In the daunorubicin-selected cell line, another, as yet uncharacterized, gene was amplified but disproportionately underexpressed. Its amplification was therefore fortuitous. We present a tentative map of the region in the hamster genome that is amplified in the multidrug-resistant cell lines which were analyzed.     

Chromosome-mediated gene transfer of hydroxyurea resistance and amplification of ribonucleotide reductase activity.

Metaphase chromosomes purified from a hydroxyurea-resistant Chinese hamster cell line were able to transform recipient wild-type cells to hydroxyurea resistance at a frequency of 10(-6). Approximately 60% of the resulting transformant clones gradually lost hydroxyurea resistance when cultivated for prolonged periods in the absence of drug. One transformant was subjected to serial selection in higher concentrations of hydroxyurea. The five cell lines generated exhibited increasing relative plating efficiency in the presence of the drug and a corresponding elevation in their cellular content of ribonucleotide reductase. The most resistant cell line had a 163-fold increase in relative plating efficiency and a 120-fold increase in enzyme activity when compared with the wild-type cell line. The highly hydroxyurea-resistant cell lines had strong electron paramagnetic resonance signals characteristic of an elevated level of the free radical present in the M2 subunit of ribonucleotide reductase. Two-dimensional electrophoresis of cell-free extracts from one of the resistant cell lines indicated that a 53,000-dalton protein was present in greatly elevated quantities when compared with the wild-type cell line. These data suggest that the hydroxyurea-resistant cell lines may contain an amplification of the gene for the M2 subunit of ribonucleotide reductase.     

Segments of Djungarian hamster chromosomes, specific for translocation of integration of amplified mdr1 genes, do not possess increased instability]

Earlier we have revealed in Djungarian hamster DM-15 cells three chromosomal segments (2p22, 5p1, 7q23-25) that are specific for transposition of amplified mdr1 genes from the site of location of resident gene copy (it was mapped to 4q21-23). In situ hybridization revealed in wild type cells no mdr1 homologous sequences in all these three segments. In this work we studied distribution of chromosomal breakages induced in DM-15 cells by aphidicolin, 5-fluorodeoxyuridine or N-phosphonacetyl-L-aspartate. The data obtained indicate that two of above mentioned segments. 2p22 and 7q23-25, contain no fragile sites. So, specificity of these segments for the transposition of amplified mdr1 genes is not due to their particular instability or to the presence in them of sequences homologous to amplifiable selectable gene.     

Structure of amplified DNA in different Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

Syrian hamster cell lines selected in multiple steps for resistance to high levels of N-(phosphonacetyl)-L-aspartate (PALA) contain many copies of the gene coding for the pyrimidine pathway enzyme CAD. Approximately 500 kilobases of additional DNA was coamplified with each copy of the CAD gene in several cell lines. To investigate its structure and organization, we cloned ca. 162 kilobases of coamplified DNA from cell line 165-28 and ca. 68 kilobases from cell line B5-4, using a screening method based solely on the greater abundance of amplified sequences in the resistant cells. Individual cloned fragments were then used to probe Southern transfers of genomic DNA from 12 different PALA-resistant mutants and the wild-type parents. A contiguous region of DNA ca. 44 kilobases long which included the CAD gene was amplified in all 12 mutants. However, the fragments cloned from 165-28 which were external to this region were not amplified in any other mutant, and the external fragments cloned from B5-4 were not amplified in two of the mutants. These results suggest that movement or major rearrangement of DNA may have accompanied some of the amplification events. We also found that different fragments were amplified to different degrees within a single mutant cell line. We conclude that the amplified DNA was not comprised of identical, tandemly arranged units. Its structure was much more complex and was different in different mutants. Several restriction fragments containing amplified sequences were found only in the DNA of the mutant cell line from which they were isolated and were not detected in DNA from wild-type cells or from any other mutant cells. These fragments contained novel joints created by rearrangement of the DNA during amplification. The cloned novel fragments hybridized only to normal fragments in every cell line examined, except for the line from which each novel fragment was isolated or the parental population for that line. This result argues that "hot spots" for forming novel joints are rare or nonexistent.     

中间偃麦草染色体2Ai-2特异PCR新标记的建立和St基因组特异序列的克隆

中间偃麦草麦、小麦和小麦－中间偃麦草2Ai－2附加系Z1、Z2、X6,代换系ZD28等进行RAPD分析,从320个RAPD引物中,鉴定出2Ai－2染色体特异的2个RAPD标记OPO05650和OPMO414000。利用这2个特异OPO05和OPM04,PCR扩增普通小麦CS（ABD）及其近缘植物中间偃麦草（E1E2St）、拟鹅冠草（St）,长穗偃麦草（E）、簇毛麦（V）、黑麦（R）、大麦（H）粗山羊草（D）等基因组DNA。结果表明,OPO05650和OPO41400均是2Ai－2染色体上St基因组区域的特异标记。将上棕2个特异片段分离回收、克隆、测序,根据测序结果重新设计、合成特异引物,成功地转换RAPD标记为SCAR（sequence characterizked amplifed region）标记SC－05和SC－M4。利用SCAR标记对不同材料进行分析的结果表明,凡含有2Ai－2染色体的抗黄矮病材料及拟鹅冠草均产生一条扩增带,不含2Ai－2染色体的材料,包括小麦、长穗麦草、簇毛麦、黑麦、在麦、粗山羊草以有含有其他他中间偃麦草染色休的附加系,均没有扩增产物,说明上棕2个SCAR标记是中间偃麦草2Ai－2染色体的特异性PCR标记,且是2Ai－2染色体上St基因组区域的特异性标记。克隆与鉴定中间偃麦草的2个SCAR扩增片段TiSCO5和TiSCM4。结果表明,克隆的中间偃麦草TiSCO5和TiSCM4特异片段,分别是St基因组特异性的寡拷贝序列有多拷贝重复序列,为St基因组遗传研究的新探针。     

Fine structure analysis of the Chinese hamster AS gene encoding asparagine synthetase

Overlapping cDNAs for Chinese hamster ovary (CHO) asparagine synthetase (AS) were isolated from a library prepared from an AS-overproducing cell line. The sequence was determined and shown to contain an open reading frame encoding a protein of Mr 64,300. The predicted amino acid sequence for the CHO AS enzyme was compared to that of the human AS enzyme and found to be 95% homologous. A potential glutamine amide transfer domain, with sequence similarity to amidotransferases from bacteria and yeast, was identified in the N-terminal portion of the protein. The cDNAs were used to screen a library of phage containing wild type CHO DNA and the genomic AS sequences were detected on three overlapping phages. Determination of the fine structural organization showed that the CHO AS gene spanned 19 kilobases and was composed of 12 exons, three of which contained the glutamine amidotransferase domain. The 5' flanking sequences were highly G + C-rich and, like other housekeeping genes, lacked TATA and CAAT boxes.     

大仓鼠DNA指纹谱探针的筛选

采用一种简便提取高质量DNA 的方法, 从大仓鼠肝脏组织中提取其总DNA , 分别以人工合成的微卫星核心序列(GTG)₅和(CA)₈做单一引物, 进行特异引物PCR 反应。电泳检测后回收15 条特异性片段。与被标记过的大仓鼠基因组DNA 反向杂交结果表明, 15 个片段中(GTG)_5-8 、(CA)_8-1b和(CA_{) 8-5b}产生了较强的阳性信号。我们依据3 个片段的测序结果设计适合DIG标记的探针, 该探针得到的大仓鼠不同地理种群个体的指纹图谱有较高的个体特异性和种群多态性, 而且与传统的来源于其它生物重复序列的探针如33.6 和33.15 形成的指纹图谱相比得到的变异适中, 便于统计。     

Functional consequences of haem orientational disorder in sperm-whale and yellow-fin-tuna myoglobins.

With the use of the isoschizomeric restriction endonucleases HpaII and MspI, we found that mouse tumour ornithine decarboxylase (ODC; EC 4.1.1.17) genes are extensively methylated. ODC genes in L1210 mouse leukaemia cells were apparently more methylated than in Ehrlich ascites carcinoma, as revealed by the use of HpaII endonuclease, yet the digestion of genomic DNA isolated from these two murine tumour cell lines with MspI, which cleaves at a CCGG sequence, also with internally methylated cytosine, resulted in an apparently identical restriction pattern. It is possible that the amplification of ODC genes in Ehrlich ascites-carcinoma cells in response to 2-difluoromethylornithine (DFMO) was associated with hypomethylation, or that less-methylated genes were amplified. A human myeloma (Sultan) cell line only revealed three separate hybridization signals when cleaved with HpaII. One of these signals was amplified under the pressure of DFMO. When cleaved with MspI, these three HpaII fragments disappeared and were replaced by a double signal of 2.3-2.4 kilobase-pairs (kbp) in size. The amplified ODC sequences in the Sultan myeloma cell line apparently originated from chromosome 2, as indicated by a unique hybridization signal in a 5.8 kbp HindIII fragment specific for the human ODC locus on chromosome 2. A comparison of different human cells, the Sultan myeloma, a lymphocytic B-cell leukaemia (Ball), normal mononuclear leucocytes and leucocytes obtained from leukaemia patients, revealed interesting differences in the methylation of ODC genes. The use of two restriction endonucleases (HpaII and CfoI), the cleavage site for both of which contains a CG sequence and which only cleave when cytosine is unmethylated, indicated that ODC genes in the lymphocytic leukaemia cells were much less methylated than those in the normal leucocytes or in the Sultan cells.     

Syntenic conservation of HSP70 genes in cattle and humans

A phage library of bovine genomic DNA was screened for hybridization with a human HSP70 cDNA probe, and 21 positive plaques were identified and isolated. Restriction mapping and blot hybridization analysis of DNA from the recombinant plaques demonstrated that the cloned DNAs were derived from three different regions of the bovine genome. One region contains two tandemly arrayed HSP70 sequences, designated HSP70-1 and HSP70-2, separated by approximately 8 kb of DNA. Single HSP70 sequences, designated HSP70-3 and HSP70-4, were found in two other genomic regions. Locus-specific probes of unique flanking sequences from representative HSP70 clones were hybridized to restriction endonuclease-digested DNA from bovine-hamster and bovine-mouse somatic cell hybrid panels to determine the chromosomal location of the HSP70 sequences. The probe for the tandemly arrayed HSP70-1 and HSP70-2 sequences mapped to bovine chromosome 23, syntenic with glyoxalase 1, 21 steroid hydroxylase, and major histocompatibility class I loci. HSP70-3 sequences mapped to bovine chromosome 10, syntenic with nucleoside phosphorylase and murine osteosarcoma viral oncogene (v-fos), and HSP70-4 mapped to bovine syntenic group U6, syntenic with amylase 1 and phosphoglucomutase 1. On the basis of these data, we propose that bovine HSP70-1,2 are homologous to human HSPA1 and HSPA1L on chromosome 6p21.3, bovine HSP70-3 is the homolog of an unnamed human HSP70 gene on chromosome 14q22-q24, and bovine HSP70-4 is homologous to one of the human HSPA-6,-7 genes on chromosome 1.     

Localization of DNA Sequences Tightly Associated with the Synaptonemal Complex in Compositional Fractions of the Golden Hamster Genome*

Synaptonemal complex (SC) isolated from spermatocyte nuclei after their exhaustive hydrolysis by DNase II contains DNA sequences tightly associated with it (SCAR DNA). Here, the compositional properties of a cloned family of golden hamster SCAR DNA were studied. For this purpose, 27 SCAR DNA clones were hybridized with compositionally fractionated golden hamster genomic DNA. The sequences of the SCAR DNA family were mainly localized in the GC-poor isochore families L1 and L2, which accounted for 63% of hybridization signals. The remaining 37% of signals pertained to the GC-rich isochore families H1 and H2. Thus, SCAR DNA proved to be distributed throughout the genome, irrespective of differences in density and sequence type between isochore families. Moreover, the SCAR DNA sequences containing the regions of homology with LINE/SINE repeats were found in all the isochore families. The compositional localization of SCAR DNA is in agreement with the hypothesis that the SC and SCAR DNA participate in chromatin reorganization during meiosis prophase I, which should result in the attachment of chromatin loops to the lateral elements of SC throughout its length.     

人体基因组随机单拷贝顺序的分离及其在染色体上的定位

本文从构建杂种细胞14-7-1的基因组文库出发,用种特异的探针分离出含有人体基因组顺序的重组子,并进一步分析了其中13个克隆,得到8个单拷贝顺序。通过与已建立的杂种细胞克隆分布板杂交以及染色体的原位杂交方法,将1个单拷贝顺序FD11-1定位在11p11-q11上。由于已经报道在11号染色体上具有3个连锁群,它们分别位于11p15、11p13和11q13上,因此,FD11-1有可能为11号染色体连锁基因图的建立提供1个有意义的座位。