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.     

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。     

癌基因Ha-ras在转化和未转化细胞中表达和调控的差异

以前的工作曾用人胃癌基因Ha-ras转化了大鼠全胚细胞系Ratl细胞,得到转化细胞Rat3-3。克隆了Ha-ras癌基因6.6kb及其上游区2.5kb DNA片段,并发现2.5kb有Alu重复顺序,说明这个片段是来源于人胃癌细胞,虽族观察到p21蛋白编码12位点突变,我们又发现转化的Rat3-3细胞的Ha-ras mRNA水平比未转化的Rat1高大约五倍;通过DNase I超敏感实验证明只有转化细胞核中的Ha-ras基因对DNaseI敏感,1μg/mL的DNaseI就有明显的降解,而未转化细胞Rat1细胞核的Ha-ras基因在15μg/mL的DNaseI中也未发现有任何降解;另外还发现转化细胞核有一种能为Ha-ras基因上游区2.5kb特异结合的核蛋白,分子量大约35kD,此核蛋白不能与6.6kb Ha-ras基因本身结合,在未转化细胞中未发现此蛋白。从这些结果推测,癌基因Ha-ras的活化,除了点突变外,还可能存在另一条活化途径,即它的上游区可能有类似增强子的调控区。     

Cloning of the DNA BglII fragments of bacteriophage T4 in the vector plasmid pSCC31

An attempt has been made to clone six BglII fragments of T4 DNA in the range of 3.3-8.1 kb in the vector plasmid pSCC31 containing a single BglII site within the gene for endonuclease EcoRI and pL promoter of phage lambda. DNA fragments were extracted from the corresponding bands of agarose gel. The following BglII fragments were cloned: the 3.3 kb fragment No. 9 containing a portion of gene 20, the gene 21 and a portion of gene 22; the 4.2 kb fragment No. 8.1 with genes 17, 18, 19 and a portion of gene 20; the 5.2 kb fragment No. 7.1 with genes 25-29 and a portion of gene 48. In the case of the fragment No. 7.1, the recombinant plasmids pRL705 and pRL707 with different orientation of phage DNA fragment were obtained. An attempt to clone the fragments No. 8.2 (4.2 kb), No. 7.2 (5.45 kb) and No. 6 (8.1 kb) was unsuccessful and this probably indicates the presence of the genes, whose products are deleterious to the growth of bacterial cell.     

人Ha-ras基因中蛋白质特异结合DNA位点的鉴定

利用迁移率改变法和DNaseⅠ足纹法观察了Ha-ras癌基因5’端上游序列与特异结合蛋白的相互作用。用限制性内切酶XmaⅠ消化6.6kb Ha-ras基因得到约10个片段,进行3’-末端标记,与T24细胞核提取液反应,经低离子强度聚丙烯酰胺凝胶电泳,发现与蛋白质特异结合的416bp片段,位于Ha-ras基因5’-端上游1230—1646区域内,靠近转录起始点1660bp处(CAP位置)。另一个与蛋白质特异结合的389bp片段,位于转录起始点上游162—551处。     

Interaction of zinc ions with arsanilazotyrosine-248 carboxypeptidase A

The interaction between arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn]) and excess zinc ions has been studied by stopped-flow and spectrophotometric methods at pH 8.2 and 7.7, I = 0.5 M (NaCl), and 25 degrees C. When excess zinc ions bind to arsanilazotyrosine-248 carboxypeptidase A, the characteristic red color, which arises from the intramolecular complex of the arsanilazotyrosine-248 residue with the active site zinc of the enzyme, changes to yellow with the inhibition of peptidase activity of the enzyme. Excess zinc ions have two binding sites for arsanilazotyrosine-248 carboxypeptidase A, and the binding constants of the first site (3.9 X 10(5) M-1 at pH 8.2; 7.1 X 10(4) M-1 at pH 7.7) are much larger than those of the second site (1.8 X 10(3) M-1 at pH 8.2; 7 X 10(2) M-1 at pH 7.7). The binding of excess zinc ions to the first site is completely correlated with the inhibition of the enzyme peptidase activity and the color change of the enzyme. The results can be understood in terms of zinc ions reacting with only one of three conformational states of arsanilazotyrosine-248 carboxypeptidase A [Harrison, L. W., Auld, D. S., & Vallee, B. L. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 4356].(ABSTRACT TRUNCATED AT 250 WORDS)     

人胃癌Ha-ras基因及上游区片段甲基化对转化效率及表达的影响

 本文将克隆于pBR322的人胃癌Ha-ras基因(PGC6.6)和带有上游区片段的Ha-ras基因(PGC9.1)的CC~*GG位点甲基化后,转化NIH3T3细胞。发现pGC6.6甲基化与非甲基化对转化效率无明显影响,而pGC9.1甲基化后转化效率明显低于非甲基化pGC9.1者,甲基化/非甲基化pGC9.1的转化效率均明显高于甲基化/非甲基化pGC6.6者。本文又对人胃癌组织及癌旁组织DNA中Ha-ras基因的HpaⅡ、Msp Ⅰ限制性内切酶图谱作了比较,并同对比较了癌及癌旁组织中Ha-ras基因的mRNA水平,发现一例病人癌组织中Ha-ras基因的CC~*GG位点甲基化程度较癌旁组织中者低,且该例中Ha-ras基因表达水平在癌组织中明显地高。这些结果,结合我们以前的研究表明:在人胃Ha-ras癌基因上游区可能存在一增强子样作用的区域,对Ha-ras基因起调控作用。该上游区CC~*GG位点的甲基化能降低这种调控作用。仅Ha-ras结构基因的CC~*GG位点甲基化不足以明显影响其转化活性。在体内,Ha-ras基因甲基化水平降低可能与其达表水平升高以至诱发癌症有关。     

Characterization of Muscarinic Autoreceptors in the Rabbit Hippocampus and Caudate Nucleus

Oxotremorine-induced inhibition of electrically evoked release of ³H-acetylcholine from brain slices preincubated with ³H-choline was used to characterize muscarinic autoreceptors in rabbit hippocampus and caudate nucleus. From the shifts to the right of the concentration-response curves of oxotremorine in the presence of muscarinic receptor antagonists, the following pKB values [95% C.I.] were determined in the hippocampus: tripinamide: 8.7 [8.5, 8.8]; himbacine: 8.4 [8.3, 8.5]; AQ-RA 741: 8.3 [8.2, 8.5]; 4-DAMP: 8.2 [8.0, 8.3]; hexahydrosiladifenidol: 7.4 [7.2, 7.5]; AF-DX 116: 7.3 [7.1, 7.4]; pirenzepine: 6.8 [6.6, 7.0]; and PD102807: 6.3 [6.0, 6.5]. In the caudate nucleus: tripinamide: 9.1 [8.9, 9.2]; 4-DAMP: 8.3 [8.2, 8.5]; himbacine: 8.1 [8.0, 8.2]; AQ-RA 741: 8.1 [8.0, 8.3]; hexahydrosiladifenidol: 7.3 [7.2, 7.4]; AF-DX 116: 7.1 [7.0, 7.2]; pirenzepine: 6.7 [6.6, 6.8]; and PD102807: 6.5 [6.2, 6.8]. These pK_B values fit best to literature values for M₂ receptors, suggesting that the muscarinic autoreceptor of the rabbit hippocampus and caudate nucleus is the m₂ gene product.     

Differences in the nuclease sensitivity between the two alleles of the immunoglobulin kappa light chain genes in mouse liver and myeloma nuclei.

In mouse myeloma T the productive kappa light chain gene differs from its aberrantly rearranged allele in the patterns of DNAase I hypersensitive sites. In the region of the alleles where they are identical in sequence they have one site in common which lies 0.8 kb downstream of the coding region; but two sites upstream of and within the C gene segment (2) are found only on the non-productive allele. Within the region of different sequences both alleles have analogously located DNAase I hypersensitive sites; they lie 0.15 kb upstream of the respective leader segments and cover putative promoter sequences. Only one of the six DNAase I hypersensitive sites is also very sensitive towards micrococcal nuclease due to its particular DNA sequence. The non-rearranged gene studied in liver nuclei has no DNAase I hypersensitive sites but is preferentially cleaved in A/T rich regions.     

Hitchhiking and linkage disequilibrium between hemoglobin S and nearby restriction sites

The association of the hemoglobin S mutation with an unusual 13 kb Hpa I restriction fragment has been reexamined using new data and a stochastic model for linkage disequilibrium. In agreement with earlier analyses with deterministic models, the present-day association of the beta S allele with the 13 kb fragment is lower than would be expected if all beta S alleles carried by American blacks were derived from a unique mutation that occurred in the ancestral Bantu population. However, from the stochastic model, if the effective population size was small for an appreciable period during the time that selection has been operating on HbS, then the confidence limits on the allelic association are very large and a unique West African beta S mutation cannot be ruled out from the two locus data alone.     

Transformation-derived Neisseria gonorrhoeae plasmids with altered structure and function.

Plasmid deoxyribonucleic acid from Neisseria gonorrhoeae containing a 7.1-kilobase (kb) (4.7-megadalton) penicillinase (Pcr) plasmid transformed homogenic gonococci to penicillinase production at a low frequency. About 25% of the penicillinase-producing gonococcal transformants contained Pcr plasmids which were either larger or smaller than the 7.1 kb donor plasmid; these Pcr plasmids varied in size from 3.45 to 42 kb. Some of these altered plasmids differed from the donor plasmid in stability or in frequency of mobilization by a 36-kb (24-megadalton) conjugative plasmid. A restriction endonuclease cleavage map of the 7.1-kilobase Pcr plasmid and several of the smaller deleted plasmids was constructed. The most common size of altered Pcr plasmid was 5.1 kb (3.4 megadaltons). A Pcr plasmid isolated from a gonococcus in London, England, was identical with these 5.1-kb transformant plasmids in both size and restriction endonuclease cleavage profiles, suggesting that the 5.1-kb Pcr plasmid could have arisen from a 7.1-kb Pcr plasmid by a transformation-associated deletion in nature.     

Stable length polymorphism of up to 260 kb at the tip of the short arm of human chromosome 16

We have completed a long-range restriction map of the terminal region of the short arm of human chromosome 16 (16p13.3) by physically linking a distal genetic locus (alpha-globin) with two recently isolated probes to telomere-associated repeats (TelBam3.4 and TelBam-11). Comparison of 47 chromosomes has revealed major polymorphic length variation in this region: we have identified three alleles in which the alpha-globin genes lie 170 kb, 350 kb, or 430 kb from the telemere. The two most common alleles contain different terminal segments, starting 145 kb distal to the alpha-globin genes. Beyond this boundary these alleles are nonhomologous, yet each contains sequences related to other (different) chromosome termini. This chromosome size polymorphism has probably arisen by occasional exchanges between the subtelomeric regions of nonhomologous chromosomes; analogous length variation is likely to be present at other human telomeres.     

Genus specificity and extensive methylation of the W chromosome-specific repetitive DNA sequences from the domestic fowl,Gallus gallus domesticus

Two female-specific repeating DNA units of 0.6 kilobase pairs (kb) and 1.1 kb, produced by digesting the genomic DNA of the White Leghorn chicken with Xho I, were cloned by inserting them into the Xho I site of an Escherichia coli plasmid vector pACYC177. Two such recombinant plasmids, pAGD0601 and pAGD1101, containing a single 0.6-kb and 1.1-kb sequence, respectively, were used as molecular probes. In situ hybridization of the ³Hprobes to the metaphase chromosomes from the female White Leghorn embryos revealed their localization in the W chromosome. Semiquantitative Southern blot hybridization with ³²P-probes in excess indicated that the 0.6-kb unit and 1.1-kb unit were repeated approximately 14,000 and 6,000 times, respectively, in the W chromosome. The two units comprised about 46% of the W chromosomal DNA. These two repeating units were found in the female genomes of every line of Gallus g. domesticus tested and in the female genomes of three jungle fowl species (G. gallus, G. sonneratii, and G. varius) but not in three species belonging to other genera in the suborder Galli. Hha I sites in the 0.6-kb and 1.1-kb repeating units were shown to be extensively methylated and a significant fraction of the Hpa II sites in the 0.6-kb repeating units were also shown to be methylated in the female genome of the White Leghorn. Methylation patterns of Hpa II sites in or around the 0.6-kb repeating units examined by the Msp I digestion were similar in the various lines of domestic fowls and the two species of jungle fowls, but G. varius (black or green jungle fowl) produced a different pattern of digestion with Msp I.     

The apolipoprotein (a) gene: a transcribed hypervariable locus controlling plasma lipoprotein (a) concentration

Lipoprotein(a) [Lp(a)] is a quantitative trait in human plasma. Lp(a) consists of a low-density lipoprotein and the plasminogen-related apolipoprotein(a) [apo(a)]. The apo(a) gene determines a size polymorphism of the protein, which is related to Lp(a) levels in plasma. In an attempt to gain a deeper insight into the genetic architecture of this risk factor for coronary heart disease, we have investigated the basis of the apo(a) size polymorphism by pulsed field gel electrophoresis of genomic DNA employing various restriction enzymes (SwaI, KpnI, KspI, SfiI, NotI) and an apo(a) kringle-IV-specific probe. All enzymes detected the same size polymorphism in the kringle IV repeat domain of apo(a). With KpnI, 26 different alleles were identified among 156 unrelated subjects; these alleles ranged in size from 32kb to 189kb and differed by increments of 5.6kb, corresponding to one kringle IV unit. There was a perfect match between the size of the apo(a) DNA phenotypes and the size of apo(a) isoforms in plasma. The apo(a) DNA polymorphism was further used to estimate the magnitude of the apo(a) gene effect on Lp(a) levels by a sib-pair comparison approach based on 253 sib-pairs from 64 families. Intra-class correlation of log-transformed Lp(a) levels was high in sib-pairs sharing both parental alleles (r = 0.91), significant in those with one common allele (r = 0.31), and absent in those with no parental allele in common (r = 0.12). The data show that the intra-individual variability in Lp(a) levels is almost entirely explained by variation at the apo(a) locus but that only a fraction (46%) is explained by the DNA size polymorphism. This suggests further heterogeneity relating to Lp(a) levels in the apo(a) gene.     

Analysis of copy number variation in the normal human population within a region containing complex segmental duplications on 22q11 using high-resolution array-CGH

A previously detected copy number polymorphism (Ep CNP) in patients affected with neuroectodermal tumors led us to investigate its frequency and length in the normal population. For this purpose, a program called Sequence Allocator was developed and applied for the construction of an array that consisted of unique and duplicated fragments, allowing the assessment of copy number variation within regions of segmental duplications. The average resolution of this array was 11 kb and we determined the size of the Ep CNP to be 290 kb. Analysis of normal controls identified 7.7 and 7.1% gains in peripheral blood and lymphoblastoid cell line (LCL) DNA, respectively, while deletions were found only in the LCL group (7.1%). This array platform allows the detection of DNA copy number variation within regions of pronounced genomic complexity, which constitutes an improvement over available technologies.