中国人胃癌和正常组织基因组DNA中癌基因Ha-ras的限制性片段长度多态性研究

本文报道以PHS-49为探针,分析了中国人群中36例胃癌患者癌组织和25位正常人体组织基因组DNA中Ha-ras基因的BamHI限制性片段长度多态性(RFLPs)。发现了10种不同长度的片段和18种基因型。其中4种小于6kb的BamHI片段是迄今国外未见报道的,这可能是中国人群遗传多态性的一个特征。此外,在胃癌组织中发现Ha-ras的一些稀有等位基因和基因型的频率明显高于正常人群。对两名胃癌患者家系中的11名成员也作了RFLPs分析,发现有些成员出现3条和4条限制性片段的杂合个体,表明这些个体的染色体上含有的Ha-ras基因不只一份拷贝。对上述现象的可能原因作了分析和讨论。     

Polymorphism of the human vitronectin gene causes vitronectin blood type.

Human blood plasma/sera are classified into three distinct vitronectin types based on the relative amount of the 75 kDa polypeptide to its cleavage product of 65 kDa. We asked whether the vitronectin blood types correlated with the polymorphism of the vitronectin gene. A portion of the vitronectin gene was amplified by using polymerase chain reaction and digested with a restriction enzyme PmaC I which may distinguish the base sequence causing the polymorphic change at the amino acid position 381. Amplified DNAs of the blood type I (75 kDa-rich), II (75/65 kDa-even), and III (65 kDa-rich) were shown to be resistant, moderately sensitive and completely sensitive to PmaC I, respectively. These results suggest that Thr at position 381 is essential for the cleavage of the vitronectin 75 kDa polypeptide and that three possible combinations of two codominant alleles of vitronectin determine three vitronectin blood types.     

Identification of caseins in goat milk

The importance of goat milk in infant diet is growing, because it is reported that goat's milk in some cases is less allergenic than cow's milk. This is due probably to the lower presence of caseins associated with a specific type of alpha(s1)-casein. In caprine breeds, four types of alpha(s1)-casein alleles are identified and associated with various amounts of this protein in milk. The contribution of strong alleles to the goat milk is approximately 3.6 g/L of alpha(s1)-casein, while for middle alleles is only 1.6 g/L, weak alleles 0.6 g/L. The contribution of null allele is very low (or non-existent). The quantity of total caseins in caprine milk is positively correlated with the amount of alpha(s1)-casein. Milk from animals possessing strong alleles contain significantly more total caseins than milk from animals without those alleles. This is important because animals with mild alleles can be employed to produce milk for allergic subjects while the other animals can be used to produce milk for the dairy industry. This work shows casein profiles of two types of classified goat milk (B, strong alpha(s1) allele, 0, null alpha(s1) allele) with two-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and it confirms the different polymorphisms at locus alpha(s1) casein.     

Selective allele loss and interference between cauliflower mosaic virus DNAs

Summary Some cauliflower mosaic virus (CaMV) alleles are selectively lost during growth of the virus in mixedly infected turnip plants. Viral DNA from plants co-inoculated with DNA of the cabbage S isolate and infectious cabbage S DNA with an extra EcoRI restriciion site lacked the extra site. The EcoRI allele was also lost in most plants co-inoculated with a non-infectious mutant of cabbage S DNA while little selective allele loss was observed with two other non-infectious mutant DNAs. Plants co-inoculated with DNAs of closely-related isolates (CM4-184 and W) contained both parental viral DNAs and some DNAs with characteristics of both parents. Interference, scored as a reduced frequency of infection or a delay in symptom appearance relative to plants inoculated with wild-type DNA, occurred when plants were inoculated with wild-type and mutant DNAs covalently attached to one another in partial dimer plasmid DNAs. Similarities in the conditions leading to selective allele loss and those leading to interference suggest that both may have been due to active gene conversion between CaMV DNA molecules.     

Analysis of the association of inherited predisposition to breast cancer with c-Ha-ras-1 oncogene alleles

Polymorphism of the human c-Ha-ras-1 gene has been analysed in 66 BamHI restricted DNAs from blood of 35 patients with "inherited" breast cancer, 7 fibroadenoma patients, 13 healthy first-degree relatives and 11 unaffected controls. Two "common" and four "unusual" alleles were detected. The frequency of "common" (6.6 and 7.4 kb) and "unusual" (6.9 kb) alleles was identical to that in the control and unaffected groups (65.8, 17.1 and 7.1%). Rare alleles (7.6 and 7.8 kb) were only detected in breast cancer patients and in healthy first-degree relatives. A 8.0 kb allele specific for control patients was also detected. No absolute relationship between the genetic predisposition to breast cancer and the Ha-ras genotype was assumed.     

DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication.

We have analysed DNA from African cassava mosaic virus (ACMV)-infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and detected ACMV-specific DNAs by blot-hybridisation. ACMV DNA forms including the previously characterised single-stranded, open-circular, linear and supercoiled DNAs along with five previously uncharacterised heterogeneous DNAs (H1-H5) were resolved. The heterogeneous DNAs were characterised by their chromatographic properties on BND-cellulose and their ability to hybridise to strand-specific and double-stranded probes. The data suggest a rolling circle mechanism of DNA replication, based on the sizes and strand specificity of the heterogeneous single-stranded DNA forms and their electrophoretic properties in relation to genome length single-stranded DNAs. Second-strand synthesis on a single-stranded virus-sense template is evident from the position of heterogeneous subgenomic complementary-sense DNA (H3) associated with genome-length virus-sense template (VT) DNA. The position of heterogeneous virus-sense DNA (H5), ranging in size from one to two genome lengths, is consistent with its association with genome-length complementary-sense template (CT) DNA, reflecting virus-sense strand displacement during replication from a double-stranded intermediate. The absence of subgenomic complementary-sense DNA associated with the displaced virus-sense strand suggests that replication proceeds via an obligate single-stranded intermediate. The other species of heterogeneous DNAs comprised concatemeric single-stranded virus-sense DNA (H4), and double-stranded or partially single-stranded DNA (H1 and H2).     

Heteroduplex molecules formed between allelic sequences cause nonparental RAPD bands.

Primers (10-mers) of random sequence were used to amplify RAPD bands from genomic DNA of an F1 strain of flax rust (Melampsora lini) and its two parent strains. One primer out of 160 tested was unusual in that it amplified a product from F1 DNA that was not amplified from either parental DNAs. The same primer also generated two RAPD bands that segregated as codominant alleles amongst F2 progeny. The nonparental band was only generated from DNAs of F2 individuals that were heterozygous for these two allelic sequences. Sequence analysis of the two RAPD alleles demonstrated greater than 99% sequence identity, although the larger allele possessed an additional 38bp relative to the smaller. Mixing of the two allelic sequences followed by denaturation and annealing in the absence of polymerase activity resulted in the formation of the nonparental band. Thus the nonparental band present in some RAPD reactions consisted of a heteroduplex molecule formed between two allelic sequences of different size. These data demonstrate that heteroduplex molecules formed between allelic RAPD products are a potential source of artifactual polymorphism that can arise during RAPD analysis.     

西瓜DUS测试标准品种SSR指纹图谱构建及应用

本研究采用代表最大限度西瓜遗传多样性的SSR核心引物组合,分析了西瓜DUS测试指南中的24份标准品种遗传多样性与核酸指纹。以基于重测序获得的SNP标记构建的17份西瓜材料的系统发育树为参照,对24份标准品种进行了遗传多样性分析,在遗传相似系数0.80处将24份标准品种分为3大类群,分析表明：核酸指纹分类比传统形态学分类更为准确。采用二维（QR）编码构建了西瓜24份标准品种的SSR指纹图谱,并利用本技术以保护品种“京欣2号”与对照品种“京欣1号”为例,进行了DUS分子鉴定测试,共扩增32个SSR位点,“京欣1号”和“京欣2号”之间存在4个位点的差异,品种间遗传相似系数为0.89,比形态学鉴定的差异位点更多且更准。本研究建立的西瓜DUS标准品种SSR指纹图谱与分子检测技术,可以应用到西瓜品种DUS分子检测实践,同时也为西瓜品种纯度与真实性鉴定及遗传背景分析提供了技术方案。     

Inheritance of RFLP loci in a loblolly pine three-generation pedigree

Summary A high-density restriction fragment length polymorphism (RFLP) linkage map is being constructed for loblolly pine (Pinus taeda L.). Loblolly pine cDNA and genomic DNA clones were used as probes in hybridizations to genomic DNAs prepared from grandparents, parents, and progeny of a three-generation outbred pedigree. Approximately 200 probes were evaluated for their ability to detect polymorphic loci between DNAs prepared from the two parent trees, 20–1010 and 11–1060, and cut with four different restriction enzymes: BamHI, DraI, EcoRI, and HindIII. More than half of the probes detecting single- or low-copy number sequences (56%) revealed polymorphisms between the two parents with at least one restriction enzyme. If necessary, an additional hybridization to DNAs prepared from the four grandparent trees was conducted to determine the zygosity of parent trees. Ten of these probes were hybridized to progeny DNAs from this cross and, as expected, the markers were inherited as simple codominant Mendelian alleles. Four of the ten probes detected segregation of three alleles at one locus, and four probes detected more than one independently segregating locus. RFLPs can be used immediately to assess genetic diversity in conifer populations and to efingerprint genotypes in tree improvement programs.     

Effect of ICRF-193, a novel DNA topoisomerase II inhibitor, on simian virus 40 DNA and chromosome replication in vitro.

The effect of ICRF-193, a noncleavable-complex-forming topoisomerase II inhibitor, on simian virus 40 (SV40) DNA and SV40 chromosome replication was examined by using an in vitro replication system composed of HeLa cell extracts and SV40 T antigen. Unlike the topoisomerase inhibitors VP-16 and camptothecin, ICRF-193 had little effect on DNA chain elongation during SV40 DNA replication, but high-molecular-weight DNAs instead of segregated monomer DNAs accumulated as major products. Analysis of the high-molecular-weight DNAs by two-dimensional gel electrophoresis revealed that they consisted of catenated dimers and late Cairns-type DNAs. Incubation of the replicated DNA with topoisomerase II resulted in conversion of the catenated dimers to monomer DNAs. These results indicate that ICRF-193 induces accumulation of catenated dimers and late Cairns-type DNAs by blocking the decatenating and relaxing activities of topoisomerase II in the late stage of SV40 DNA replication. In contrast, DNA replication of SV40 chromosomes was severely blocked by ICRF-193 at the late stage, and no catenated dimers were synthesized. These results are consistent with the finding that topoisomerase II is required for unwinding of the final duplex DNA in the late stage of SV40 chromosome replication in vitro.     

Oncovirus-induced permanent genetic instability in Drosophila melanogaster

Mutant alleles of a system of genetic instability induced by oncoviral DNAs were shown to demonstrate an unstable manifestation 500 generations after their emergence. A cytogenetic analysis of oncovirus-induced unstable lines has revealed numerous chromosome rearrangements. For the Lobe alleles of this system, a specific chromosome rearrangement, Df(2L) = 35C-36B, was found on the left arm of chromosome 2. We used recessive lethal mutations involving DNA rearrangements in a successful construction of cross systems for "explosive" instability.     

中国人COL2A1基因座的扩增片段长度多态性

用聚合酶链式反应、高分辨聚丙烯酰胺凝胶水平电泳及银染技术对位于人类Ⅱ型胶原基因终止密码下游非转录区１．３ｋｂ处的可主数目串联重复育列进行了研究。制备了由人类不同基因型ＤＮＡ混合而成的人类等位基因型参考物，根据实验结果进行了命名。     

The ''cation-dependent'' mannose 6-phosphate receptor binds ligands in the absence of divalent cations

EcoRI restriction fragment length polymorphisms (RFLP) at the human c-mos locus were analysed in DNAs of normal individuals and tumour patients. Two alleles with fragment lengths of 2.6 kb (A1) and 5.6 kb (A2) respectively were detected. The allele distribution among the tumour group was similar to that of the control group. No difference was found between the allele frequencies in leucocytes and tumour tissue DNA of the same patients.     

Two-step multiplex polymerase chain reaction improves the speed and accuracy of genotyping using DNA from noninvasive and museum samples

Many studies in molecular ecology rely upon the genotyping of large numbers of low‐quantity DNA extracts derived from noninvasive or museum specimens. To overcome low amplification success rates and avoid genotyping errors such as allelic dropout and false alleles, multiple polymerase chain reaction (PCR) replicates for each sample are typically used. Recently, two‐step multiplex procedures have been introduced which drastically increase the success rate and efficiency of genotyping. However, controversy still exists concerning the amount of replication needed for suitable control of error. Here we describe the use of a two‐step multiplex PCR procedure that allows rapid genotyping using at least 19 different microsatellite loci. We applied this approach to quantified amounts of noninvasive DNAs from western chimpanzee, western gorilla, mountain gorilla and black and white colobus faecal samples, as well as to DNA from ~100‐year‐old gorilla teeth from museums. Analysis of over 45 000 PCRs revealed average success rates of > 90% using faecal DNAs and 74% using museum specimen DNAs. Average allelic dropout rates were substantially reduced compared to those obtained using conventional singleplex PCR protocols, and reliable genotyping using low (< 25 pg) amounts of template DNA was possible. However, four to five replicates of apparently homozygous results are needed to avoid allelic dropout when using the lowest concentration DNAs (< 50 pg/reaction), suggesting that use of protocols allowing routine acceptance of homozygous genotypes after as few as three replicates may lead to unanticipated errors when applied to low‐concentration DNAs.     

Localization of the cryptdin locus on mouse chromosome 8

Cryptdin is a defensin-related peptide, and its mRNA accumulates to high abundance in epithelial cells of intestinal crypts beginning in the second week of postnatal development. The cryptdin (Defcr) locus was assigned to mouse chromosome 8 by Southern blotting of DNAs from mouse/hamster somatic hybrid cell lines. Analysis of somatic hybrid DNAs for mouse-specific restriction fragments showed zero discordance and perfect concordance with chromosome 8. The Defcr locus was localized on chromosome 8 by analysis of DNAs from recombinant inbred (RI) strains of mice after identification of three potential Defcr alleles based on restriction fragment length polymorphisms (RFLPs) in inbred strains. The strain distribution patterns of the Defcr locus were compared with those of chromosome 8 markers in five panels of RI strains. Analysis of cosegregation of Defcr with xenotropic proviral locus Xmv-26 and additional loci confirmed the chromosomal assignment and showed that Defcr is on proximal chromosome 8 within approximately 6 (1.3 to 21.3) cM of Xmv-26. The mouse Defcr locus and the human defensin gene(s) located on chromosome 8p23 appear to map to homologous regions.     

Sequence variations at a complex microsatellite locus in rice and its conservation in cereals

In an attempt to study changes associated with microsatellites in rice, the DNAs of cultivated rice, including indica and japonica varieties, and wild rice genotypes were amplified by the polymerase chain reaction with primers flanking the (GATA) _n and (AC) _n repeats at a microsatellite-containing locus OS1E6 (Genebank accession number AFO16647) previously reported from a PstI rice (var. Malkolam) genomic library in pUC18. Eight alleles of varying sizes were obtained which were cloned and sequenced. Sequencing data indicated that the size variations of the different alleles were due to differences in the repeat number as well as to sequence variations in the region flanking the microsatellite motifs. In order to study the presence of this complex microsatellite-containing locus of rice in different cereals, their DNAs were amplified using primers flanking the OS1E6 locus. It was found that this locus was present in the various cereal genotypes analyzed, indicating its conservation across different cereal members. Received: 10 March 2000 / Accepted: 14 April 2000     

Phylogenetic relationships in the stone fruit group of Prunus as revealed by restriction fragment analysis of chloroplast DNA.

In order to clarify the genetic relationships among stone fruits, a restriction fragment analysis of chloroplast DNAs (cpDNAs) was applied to cultivated Prunus species, whose genetic diagnoses are difficult because of their heterogeneity and long life span. Chloroplast DNAs (cpDNAs) were extracted from leaves of nine stone fruit accessions covering six species of Prunus. A restriction fragment analysis was conducted by gel electrophoresis after digestion with these endonucleases. The genome sizes of the cpDNAs were about 135-139 kbp, and the fruits were classified into seven chloroplast genome types according to their restriction fragment patterns. Two peach cultivars and nectarine were found to harbor identical plastomes, differing from those of two wild peaches and the European plum. This suggests that two cultivated peaches (P. persica) did not receive the cytoplasm from the wild peaches, P. mira and P. davidiana. Phylogenetic relationships among these types were then estimated based on the shared common fragments among the species.     

Origin of rare Ha-ras alleles: relationship of VTR length to a 5' polymorphic Xho I site

Amongst the four common Ha-ras alleles in both controls and cancer patients, we detected the presence of a polymorphic Xho I site associated specifically with the 6.6 and 7.7 kb Bam HI fragments but absent from the 7.1 and 8.2 kb alleles, as recently reported by others. We have extended this study and report here, the consistent appearance of this Xho I site in unusual alleles close in size to the two common alleles of 6.6 and 7.7 kb, in control lymphoblastoid DNA samples in a variety of tumor DNAs. Unusual alleles grouped around the 7.1 and 8.2 kb common alleles on the other hand, did not possess the Xho I site. The consistent presence of the Xho I site polymorphism, in the unusual Ha-ras alleles surrounding the 6.6 and 7.7 kb common alleles and its absence in alleles around the 7.1 and 8.2 kb common alleles, suggests that the unusual ones are derived from the corresponding common alleles to which they are closest in size.