小麂Sry基因的克隆和测序

应用人的性别决定基因SRY(Sex-determining Region Y gene,SRY)中HMG框内的一对引物,对小麂细胞株的基因组DNA进行PCR扩增,得到雄性小麂细胞的220bp扩增产物,而在雌性小麂细胞中未发现扩增产物。将雄性小麂细胞的220bp扩增产物通过T-A互补法克隆到质粒pGEM-T 载体中,筛选阳性克隆进行DNA测序。测序结果表明小麂Sry基因保守序列与人的SRY基因保守区相同碱基的比值为152/184,达到82.6%。提示小麂Sry基因与人的SRY基因存在着较高的同源性,说明SRY基因在进化过程中高度保守。 Abstract:Using the primers from SRY gene——HMG Box for PCR amplification in genomic DNA of Muntiacus reevesi cell strains,a 220bp fragment was obtained in the male but not in the female.The 220bp fragment was cloned into the pGEM-T vector using T/A clone method.The identified positive clone was sequenced.The result shows that 82.6% nucleotides(152bp/184bp) are homologous between Muntiacus Sry and human SRY gene.It suggests that SRY is highly conserved during evolution.     

Sequential amplification of dihydrofolate reductase and multidrug resistance genes in Chinese hamster ovary cells selected for stepwise resistance to the lipid-soluble antifolate trimetrexate

We describe the development of resistance to trimetrexate and piritrexim (BW 301U) by a stepwise selection protocol in Chinese hamster ovary cells. Selection in trimetrexate resulted in initial resistance as a result of dihydrofolate reductase gene amplification. Several trimetrexate-resistant variants that display 250-340-fold and 25-50-fold resistance to lipophilic and hydrophilic antifolates, respectively, were established. Increased antifolate resistance was associated with a prominent overexpression of dihydrofolate reductase as determined from the elevated folate reductase activity, cellular labeling with fluorescein-methotrexate, and steady-state mRNA levels as a result of a consistent dihydrofolate reductase gene amplification. However, upon subsequent incremental increases in trimetrexate, further resistance was also associated with amplification of the multidrug resistance gene. This resulted in overexpression of P-glycoprotein and a subsequent 20-50-fold collateral resistance to pleiotropic drugs such as adriamycin, actinomycin D, vinca alkaloids, etoposide, and colchicine. In contrast, initial resistance following selection with low piritrexim concentrations resulted from an unknown mechanism(s) not involving overproduction of either dihydrofolate reductase or P-glycoprotein. This piritrexim resistance was shared with trimetrexate but not with methotrexate. Upon further selection with piritrexim, resistant variants emerge with amplified dihydrofolate reductase but not with multidrug resistance genes. These variants were subsequently resistant to both hydrophilic and lipophilic folate antagonists but retained sensitivity to pleiotropic drugs. The pattern of resistance with methotrexate, trimetrexate, and piritrexim shared a common mechanism, dihydrofolate reductase gene amplification, but differed regarding the additional amplification of the multidrug resistance gene in trimetrexate-resistant cells as well as the emergence of an additional unknown mechanism(s) of resistance to lipid-soluble antifolates upon initial selection in piritrexim.     

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.     

RAPD analysis of jasmine rice-specific genomic structure.

Total genomic DNA was extracted from 29 samples of rice seed, including jasmine rice Oryza sativa L. subsp indica 'KDML105', 'KDML105'-derived varieties, nonaromatic Thailand rice, and japonica rice. Polymorphism in RAPD profiles was analyzed to explore the genomic structure specific to jasmine rice. The degree of band sharing was used to evaluate genetic distance between varieties and to construct a phylogenetic tree. RD15, CNTLR85033, and CNT87040 were found to be closest to 'KDML105', which was consistent with the true relation among them. Four RAPD fragments that cooperatively distinguished jasmine rice from others were cloned and sequenced. PCR amplification using pairs of primers designed specifically further confirmed the credibility of the RAPD result. Comparison through Genbank revealed that a 454 bp RAPD band was similar to the first intron of a putative Cf2/Cf5 disease resistance gene and a 1,107 bp RAPD band similar to a wall-associated kinase (wak) gene sequence.     

不结球白菜细胞质雄性不育相关基因的克隆及表达

从不结球白菜CMS新种质中分离得到的一个cDNA-AFLP差异片段,采用RT-PCR和RACE技术成功克隆了一个α-微管基因的cDNA全长序列,命名为TUBA2(DDBJ登录号为AB445012)。序列分析结果表明,该基因全长1 709 bp,最大开放阅读框为1 353 bp,编码450个氨基酸序列,与已公布的α-微管基因有较高的同源性。系统进化树分析发现,该基因在不同植物间具有高度保守性。Southern杂交表明TUBA2属于不结球白菜多基因家族的一个单一克隆基因。实时定量RT-PCR检测表明,该基因在不育系中的表达量显著低于保持系,同时在不同组织和细胞减数分裂不同时期该基因的表达量也存在明显差异。     

Peptide transport by the multidrug resistance pump.

The membrane P-glycoprotein (P170) is an ATP-hydrolyzing transmembrane pump, and elevated levels of P170, due to higher expression with or without amplification of the multidrug resistance gene (mdr1), result in resistance to a variety of chemotherapeutic agents in mammalian cells. The function of the P170 pump has been proposed as a protection against toxic substances present in animal diets. Here we describe a Chinese hamster ovary cell line that was selected for resistance to a synthetic tripeptide, N-acetyl-leucyl-leucyl-norleucinal (ALLN). This ALLN-resistant variant shows the classical multidrug resistance (MDR) phenotype, including overexpression and amplification of the mdr1 gene. Additionally, a mouse embryo cell line overexpressing the transfected mdr1 gene is likewise resistant to ALLN. Our results demonstrate that P170 is capable of transporting peptides and raise the possibility that the mdr1 gene product or other MDR-like genes, present in the genome of mammalian cells, may be involved in secretion of peptides or cellular proteins as is the case with the structurally similar hylB and ste6 gene products of Escherichia coli and yeast, respectively.     

Influence of PCR parameters on amplifications of HIV-1 DNA: establishment of limiting sensitivity

We have investigated the optimal reaction conditions and the limiting sensitivity for detection of HIV-1 DNA by PCR. The amplification systems studied were gag (SK38/SK39); pol (P3/P4); and two other systems described here for the first time, LTR (LTR1/LTR2) and nef (Nef1/Nef2), which amplify fragments of 115 bp, 308 bp, 632 bp and 643 bp, respectively. Two PCR profiles were assayed, and the requirements for deoxynucleoside triphosphate and MgCl2 concentrations for each amplification reaction were determined. Optimal reaction conditions were oriented toward selecting maximal amplification of the expected size fragment. Limiting sensitivity was estimated by testing the decreasing copy number of a plasmid containing HIV-1 genome and obtaining a positive amplification signal with at least 5, 5, 10 and 5 copies for LTR, gag, pol and nef, respectively. We conclude that the establishment of the detection sensitivity on a PCR is an important parameter to be considered for the interpretation of results on HIV-1 infection.     

An altered pattern of cross-resistance in multidrug-resistant human cells results from spontaneous mutations in the mdr1 (P-glycoprotein) gene

Multidrug resistance in human cells results from increased expression of the mdr1 (P-glycoprotein) gene. Although the same gene is activated in cells selected with different drugs, multidrug-resistant cell lines can be preferentially resistant to their selecting agent. The mdr1 cDNA sequence from vinblastine-selected KB cells, which are uniformly resistant to different lipophilic drugs, was compared with the corresponding sequence from colchicine-selected KB cells preferentially resistant to colchicine. These sequences differ at three positions, resulting in a single amino acid change in P-glycoprotein. These differences result from mutations that occurred during colchicine selection. The appearance of these mutations coincides with the emergence of preferential resistance to colchicine. We have constructed biologically active mdr1 cDNA clones that express either wild-type or mutant P-glycoprotein. Multi-drug-resistant transfectants obtained with the mutant sequence were characterized by increased relative resistance to colchicine compared with transfectants obtained with wild-type sequence. mdr1 mutations are therefore responsible for preferential resistance to colchicine in multidrug-resistant KB cells.     

Sequence requirements for function of the Drosophila chorion gene locus ACE3 replicator and ori-beta origin elements

The developmentally regulated amplification of the Drosophila third chromosome chorion gene locus requires multiple chromosomal elements. Amplification control element third chromosome (ACE3) appears to function as a replicator, in that it is required in cis for the activity of nearby DNA replication origin(s). Ori-beta is the major origin in the locus, and is a sequence-specific element that is sufficient for high-level amplification in combination with ACE3. Sequence requirements for amplification were examined using a transgenic construct that was buffered from chromosomal position effects by flanking insulator elements. The parent construct supported 18- to 20-fold amplification, and contained the 320 bp ACE3, the approximately 1.2 kb S18 chorion gene and the 840 bp ori-beta. Deletion mapping of ACE3 revealed that an evolutionarily conserved 142 bp core sequence functions in amplification in this context. Several deletions had quantitative effects, suggesting that multiple, partially redundant elements comprise ACE3. S. cerevisiae ARS1 origin sequences could not substitute for ori-beta, thereby confirming the sequence specificity of ori-beta. Deletion mapping of ori-beta identified two required components: a 140 bp 5' element and a 226 bp A/T-rich 3' element called the beta-region that has significant homology to ACE3. Antibody to the origin recognition complex subunit 2 (ORC2) recognizes large foci that localize to the endogenous chorion gene loci and to active transgenic constructs at the beginning of amplification. Mutations in Orc2 itself, or the amplification trans regulator satin eliminated the ORC2 foci. By contrast, with a null mutation of chiffon (dbf4-like) that eliminates amplification, diffuse ORC2 staining was still present, but failed to localize into foci. The data suggest a novel function for the Dbf4-like chiffon protein in ORC localization. Chromosomal position effects that eliminated amplification of transgenic constructs also eliminated foci formation. However, use of the buffered vector allowed amplification of transgenic constructs to occur in the absence of detectable foci formation. Taken together, the data suggest a model in which ACE3 and ori-beta nucleate the formation of a ORC2-containing chromatin structure that spreads along the chromosome in a mechanism dependent upon chiffon.     

Identification of small gains and losses in single cells after whole genome amplification on tiling oligo arrays

Clinical DNA is often available in limited quantities requiring whole-genome amplification for subsequent genome-wide assessment of copy-number variation (CNV) by array-CGH. In pre-implantation diagnosis and analysis of micrometastases, even merely single cells are available for analysis. However, procedures allowing high-resolution analyses of CNVs from single cells well below resolution limits of conventional cytogenetics are lacking. Here, we applied amplification products of single cells and of cell pools (5 or 10 cells) from patients with developmental delay, cancer cell lines and polar bodies to various oligo tiling array platforms with a median probe spacing as high as 65 bp. Our high-resolution analyses reveal that the low amounts of template DNA do not result in a completely unbiased whole genome amplification but that stochastic amplification artifacts, which become more obvious on array platforms with tiling path resolution, cause significant noise. We implemented a new evaluation algorithm specifically for the identification of small gains and losses in such very noisy ratio profiles. Our data suggest that when assessed with sufficiently sensitive methods high-resolution oligo-arrays allow a reliable identification of CNVs as small as 500 kb in cell pools (5 or 10 cells), and of 2.6–3.0 Mb in single cells.     

Selection for high-level chloroquine resistance results in deamplification of the pfmdr1 gene and increased sensitivity to mefloquine in Plasmodium falciparum.

A chloroquine resistant cloned isolate of Plasmodium falciparum, FAC8, which carries an amplification in the pfmdr1 gene was selected for high-level chloroquine resistance, resulting in a cell line resistant to a 10-fold higher concentration of chloroquine. These cells were found to have lost the amplification in pfmdr1 and to no longer over-produce the protein product termed P-glycoprotein homologue 1 (Pgh1). The pfmdr1 gene from this highly resistant cell line was not found to encode any amino acid changes that would account for increased resistance. Verapamil, which reverses chloroquine resistance in FAC8, also reversed high-level chloroquine resistance. Furthermore, verapamil caused a biphasic reversal of chloroquine resistance as the high-level resistance was very sensitive to low amounts of verapamil. These data suggest that over-expression of the P-glycoprotein homologue is incompatible with high levels of chloroquine resistance. In order to show that these results were applicable to other chloroquine selected lines, two additional mutants were selected for resistance to high levels of chloroquine. In both cases they were found to deamplify pfmdr1. Interestingly, while the level of chloroquine resistance of these mutants increased, they became more sensitive to mefloquine. This suggests a linkage between the copy number of the pfmdr1 gene and the level of chloroquine and mefloquine resistance.     

Overexpression of P-glycoprotein in human lung carcinoma xenografts after fractionated irradiation in vivo.

In vivo exposure of a human epidermoid lung carcinoma xenograft to seven irradiation treatments of 10 Gy in consecutive passages resulted in expression of resistance to vincristine. This about threefold drug resistance was detectable with a single dose of 1 mg/kg vincristine. Characterization of the radiation-pretreated subline showed that overexpression of P-glycoprotein, as determined by immunofluorescence and Mabs C219 and 265/F4, occurred in this tumor. After six X-ray fractions, only single positive cells were observed, whereas seven fractions produced an intense immunofluorescent reaction with both antibodies. Southern blot analyses indicated that no gene amplification had occurred. This result shows that irradiation can influence expression of P-glycoprotein and in this way influences drug resistance.     

血小板生成素改构体基因的克隆和序列分析

采用PCR技术,根据文献报道的鼠TPO成熟肽基因序列,设计并合成两对引物,以鼠TPO cDNA为模板,扩增获得mTPO N端153个氨基酸的478bp cDNA片段及鼠TPO全长1032bp cDNA片段,mTPO153片段与合成的碱性成纤维生长因子序列中Lys119-Lys135as的51bp肝素结合位点DNA片段连接,克隆到M13mp18及M13mp19载体中进行双向测序;同时将扩增的鼠TPO全长cDNA片段克隆到M13mp18及M13mp19载体中进行双向测序。证明获得鼠血小板生成素与肝素结合位点基因及鼠TPO全长基因,继之以pMAL-C2X为表达载体构建表达质粒,并经PCR及酶切鉴定。     

Optimization of conditions for the polymerase chain reaction amplification of DNA from culturable and nonculturable cells of Vibrio vulnificus

Abstract: A series of 16 buffers, differing in pH and MgCl₂ concentration, were used to optimize the polymerase chain reaction (PCR) amplification of a 388 bp region of the hemolysin / cytolysin gene from cells of Vibrio vulnificus present in both the culturable and nonculturable states. Both the opaque and translucent morphotypes were examined. Using whole cell lysates, we were able to obtain amplification of DNA from as few as 28.5 cells present in the viable but nonculturable state. With one exception, all buffers that produced amplification using culturable cells also produced amplification using nonculturable cells. However, regardless of the buffer employed, 100 times more nonculturable cells than culturable cells were required to obtain a PCR product. Our data suggest that caution should be exercised when employing PCR optimized against culturable cells when this method is employed for the detection of nonculturable cells.     

Reiterated gene amplifications at specific short homology sequences in phage T4 produce Hp17 mutants

Bacteriophage T4 gene 17 amplification mutants (Hp17) selected by growth of gene 17 amber mutants on ochre suppressor strains of Escherichia coli carry two to more than sixfold tandem head-to-tail repeats of the gene 17-18 region (Wu & Black, 1987). We characterized the structures of Hp17 isolates by restriction enzyme mapping and Southern blot analysis. The left and right boundaries of the amplified sequences were mapped within genes 16 and gene 18 or 19, respectively. The TaqI-restriction fragments containing the novel junctions arising from fusion of the amplified gene were then cloned and sequenced. Three Hp17 mutants arose from rearrangement in one five base-pair (bp) block within a G + C-rich region of partial homology (24 bp with 4 mismatches) between genes 16 and 19. Moreover, an oligonucleotide probe showed that 190/191 mutants isolated had recombined within the 5 bp block, and other rearrangements within this 24 bp region were not detected. Only one anomalous Hp mutant rearranged elsewhere between genes 16 and 18 in a 14 bp homology region with one mismatch. Elimination of gene alt of phage T4 is required for isolation of Hp17 mutants, apparently because more DNA can be packaged into alt- heads. Requirements for the dispensable replication and recombination genes of T4 were probed; T4 topoisomerase (39, 52, 60), primase (58/61), and uvsX are required, whereas the host recA gene and T4 denV gene do not appear to be required for isolation of the Hp17 mutants. The evidence suggests an initiating sequence-specific rearrangement leads to the T4 Hp17 amplification mutants.