Ha-ras-1 alleles in Norwegian lung cancer patients

Summary We have examined DNA restriction fragment length polymorphisms (RFLP) of the Ha-ras-1 gene in DNA from 118 lung cancer patients and 123 unaffected controls. When DNA samples were digested with MspI/ HpaII restriction endonucleases. Southern blot analysis demonstrated 4 common, 4 intermediate and 7 different rare alleles in the combined population after hybridization to the pGDa1 probe. Six of the rare alleles were unique for the lung cancer group and 1 rare allele for the control group. The frequency of rare alleles in lung cancer patients (10/236) was significantly different (P<0.01) from the control group (1/246). The lung cancer group also had a significantly lower frequency of the common 2.57 kb fragment than the controls (P<0.02). The results thus indicate that Ha-ras genotyping may be of value in lung cancer risk assessment.     

The procollagen type III, alpha 1 (COL3A1) gene first intron expresses poly-A+ RNA corresponding to multiple ESTs and putative miRNAs

The mouse COL3A1 first intron is 9684 bp. RNA's of approximately 1.6 and 3.0 kb were detected by Northern hybridization analysis of poly‐A RNA from fetal mice and total RNA from suckling and adult mouse intestine using ³²P‐labeled, anti‐sense RNA synthesized from a mouse COL3A1 first intron, 5 prime region, 5.4 kb Xba I fragment (1655–7030 bp), recombinant plasmid (pPI5.4x). Expression of the 1.6 and 3.0 kb RNA's was significantly reduced in adult mouse intestine, indicating that these RNAs are developmentally regulated. “BLAST” analysis indicated that the mouse first intron 5 prime sequence has 94–100% identity to 13 mouse ESTs. These mouse first intron EST's lie within the 5.4 Xba I fragment of the mouse COL3A1 first intron. Two of the mouse first intron EST's have significant identity to known miRNA, mature sequences, mmu‐miR‐466f‐3P, mmu‐miR‐1187, and mmu‐miR‐574‐5P as well as others. Predicted targets for mmu‐miR‐466f‐3P include COL1A1, COL19A1, COL11A2, COL4A1, and COL4A5 indicating that COL3A1 intronic miRNAs may regulate the expression of other collagen genes in development. J. Cell. Biochem. 112: 541–547, 2011. © 2010 Wiley‐Liss, Inc.     

猪α-1,3-半乳糖转移酶基因打靶载体的构建

目的:构建猪α-1,3-半乳糖转移酶(GGTA1)基因的正负筛选打靶载体。方法:以原代猪胚胎成纤维细胞基因组DNA为模板,采用长程PCR方法扩增出GGTA1基因的2条片段;以长约2kb包含部分第9外显子的片段为同源短臂,在XbaⅠ和ClaⅠ位点插入pLoxP质粒正筛选标记neo基因的3'端;以长约5.4kb包括部分第8外显子、全部第8内含子及部分第9外显子的片段为同源长臂,于NotⅠ位点插入该质粒中neo基因的5'端;2.7kb的负筛选标记tk基因位于载体中同源短臂的3'端外侧。结果与结论:酶切、PCR及测序结果表明,同源臂被正确连接至质粒pLoxP,成功构建了猪GGTA1基因正负筛选打靶载体pSL/GT。     

Molecular cloning and expression of the Cellulomonas flavigena cell-associated amylase gene in E. coli and Cellulomonas flavigena

Summary The gene encoding the inducible cell-associated amylase activity was cloned on a 1.5kb Pst1 fragment into pUC8 in E.coli giving the recombinant plasmid pJA 871, and subcloned onto a shuttle vector, pJA85, and transferred into Cellulomonas flavigena AP1(amy^-). Expression was observed in both organisms with increased levels being observed from the recombinant in Cellulomonas compared to the parent strain. The 1.5kb fragment was reoriented in pJA871 and the same level of expression observed in both orientations. Tn1000 insertions into the cloned fragment revealed the location of the coding region. Nucleotide sequencing of both ends of the cloned fragment revealed one open reading frame preceded by a putative control region.     

Cloning of a 1.1-kb Fragment Including srnB+ Gene in the F Plasmid and Isolation of an srnB Mutant

The srnB⁺ gene, promoting stable RNA degradation at 42 C in the presence of rifampin, was cloned by using pBR322 as a vector; it was located on a 1.1-kilobase (kb) EcoRI/BamHI fragment between 1.4 and 2.5 kb of the F plasmid. The region between 93.3 and 4.0 kb of the F plasmid was physically mapped by using restriction endonucleases EcoRI, HindIII, BamHI, PstI, and SmaI, with reference to a standard HindIII site in IS3. An srnB1 mutant was isolated from a chimeric plasmid, pOY54, after treatment of its DNA with hydroxylamine. The srnB1 allele on the F fragment of the mutant plasmid was recessive to the wild-type allele. Thermal elevation of cell cultures to 39 C was high enough to promote RNA degradation in strain YS12 carrying plasmid pOY54.     

R483 and F Plasmid Genes Promoting RNA Degradation: Comparative Restriction Mapping

The gene promoting nucleic-acid degradation (pnd) on IncIa plasmid R483 was cloned into pBR322. It is located on a 0.85 kilobase (kb) EcoRI-SalI fragment and is close to Tn7. The pnd gene has similar properties to the srnB gene on the F plasmid. A cleavage map of the 0.85 kb pnd fragment was constructed and compared with that of the 1.18 kb EcoRI-BamHI fragment containing the srnB gene. These two regions showed marked heterogeneity as evidenced by their distinctly different restriction maps. This result suggests separate paths of evolution of the two genes for stable RNA degradation.     

The polymorphism of the plasma inter-α-trypsin inhibitor (ITI) and its relationship to the heavy chain H1 subunit gene (ITIH1) at 3p211-212

We investigated the ITI protein polymorphism in linkage analysis, usingDraI andSstI as restriction fragment length polymorphism (RFLP) markers for the ITIH1 gene. Isoelectric focusing (IEF) classification from 76 individual plasma samples and RFLP analysis from the corresponding DNA preparations disclosed linkage disequilibrium between the phenotypic IEF patterns of the two common ITI alleles, ITI^*1 and ITI^*2, and the diallelic DNA polymorphisms of two ITIH1 RFLPs, represented byDraI 4.0 kb andDraI 2.4 + 1.6 kb, and bySstI 6.7 kb andSstI 6.0 + 0.7 kb, for the ITI 1 and ITI 2 IEF phenotypes, respectively, and byDraI 4.0/2.4 + 1.6 kb andSstI 6.7/6.0 + 0.7 kb for the heterozygous ITI 1–2 IEF phenotype. Linked segregation between either of the RFLPs and the polymorphic ITI plasma protein locus has been established in nine informative family pedigrees. The less frequent allele in Europeans, ITI^*3, is not represented by a further allelic restriction fragment in either RFLP. The significant linkage disequilibrium observed in this genetic study indicates that the ITI locus, with the alleles ITI^*1 and ITI^*2, must be close to, or reside within, the ITIH1 gene. The diallelic ITI protein polymorphism therefore provides an informative phenotypic marker system for chromosome 3p211-212.     

Transformation of the Acidophilic Heterotroph Acidiphilium facilis by Electroporation

We constructed a cloning vector for use in the acidophilic heterotroph Acidiphilium facilis. The vector pAH1O1 (8.8kb) was constructed from a 6.1 kb restriction fragment of the Acidiphilium plasmid pAU1 and a pUC19 carrying a β-lactamase gene. The antibiotic resistance gene was efficiently expressed in A. facilis. Several factors which influenced the transformation efficiency were optimized, resulting in a transformation efficiency of up to 3 X 10³ transformants per μg of plasmid DNA at a field strength of 10kV/cm with a 7.0ms pulse.     

Identification of a centromeric activity in the autonomously replicating TRA region allows improvement of the host-vector system for Candida maltosa

A centromeric activity was identified in the previously isolated 3.8 kb DNA fragment that carries an autonomously replicating sequence (ARS) from the yeast Candida maltosa. Plasmids bearing duplicated copies of the centromeric DNA (dicentric plasmids) were physically unstable and structural rearrangements of the dicentric plasmids occurred frequently in the transformed cells. The centromeric DNA activity was dissociated from the ARS, which is 0.2 kb in size, and was delimited to a fragment at least 325 by in length. The centromeric DNA region included the consensus sequences of CDEI (centromeric DNA element I) and an AT-rich CDEII-like region of Saccharomyces cerevisiae but had no homology to the functionally critical CDEIII consensus. A plasmid bearing the whole 3.8 kb fragment was present in 1–2 copies per cell and was maintained stably even under non-selective culture conditions, while a plasmid having only the 0.2 kb ARS was unstable and accumulated to high copy numbers. The high-copy-number plasmid allowed us to overexpress a gene to a high level, which had never been attained before, under the control of both constitutive and inducible promoters in C. maltosa.     

A DNA Region That Complements on Escherichia coli cysG Mutation in Thiobacillus Ferrooxidans

Thiobacillus ferrooxidans AP19-3 has a novel NADH-dependent sulfite reductase in the periplasmic space. The gene responsible for the appearance of NADH-dependent sulfite reductase activity was cloned into a vector plasmid pBR322 to give a 5.7-kb hybrid plasmid, pTHS1, which contains a 1.3-kb DNA fragment of T. ferrooxidans AP19-3. When pTHS1 was used to transform sulfite reductase deficient E. coli mutants, strain AT2455 (cysG), JM246 (cysl), and AT2427 (cysJ), it complemented only the E. coli cysG mutation. Since cysG codes for S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase, the enzyme involved in siroheme synthesis, the results indicate that the DNA region that codes for S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase is present in a T. ferrooxidans 1.3 kb DNA fragment on pTHS1.     

Definitive RFLPs to distinguish between the human complement C4A/C4B isotypes and the major Rodgers/Chido determinants: Application to the study of C4 null alleles

Definitive restriction fragment length polymorphisms (RFLPs) representing the exact locations responsible for isotypicity between the human complement components C4A and C4B, and their generally associated major Rodgers (Rg1) and Chido (Ch1) antigenic determinants, have been designed. By means of a C4d-specific genomic probe for Southern blot analysis, a C4A gene can be defined by the presence of the 276 bp and 191 bp N 1 a IV fragments, while a C4B gene can be defined by a single 467 bp N1aIV fragment. In addition, an Rgl-expressing C4 gene can be represented by a 565 bp EcoO 109 fragment, and a Chl-expressing C4 gene by a 458 by EcoO 109 fragment, under the same conditions. All these polymorphic restriction fragments can be unambiguously and conveniently detected. In combination with the Taq I polymorphic patterns specific for the C4 loci and for the neighboring 21-hydroxylase genes, the nature and structure of the tandem C4,21-hydroxylase gene complex can be elucidated. In this study, it is inferred that the null allele of the HLA haplotype B44 DR6 C4A3 C4BQO is not a C4B allele, but probably encodes another C4A 3 allotype at the second C4 locus.Abbreviations used in this paper C4 (long) - C4 gene of 22 kb, with a 6–7 kb intron - C4 (short) - C4 gene of 16 kb, without a 6–7 kb intron; complotype SCO1, factor B S, C2 C, C4A QO; C4B 1 Dedicated to the memory of our teacher, the late Professor Rodney Porter C. H. F. R. S.     

Molecular cloning in streptococci: Physical mapping of the vehicle plasmid pSM10 and demonstration of intergroup DNA transfer

Summary The streptococcal resistance plasmid pSM10 (8.3 kb), a deletion derivative of pSM10419 (22.9 kb) determining constitutive erythromycin and lincomycin resistance, was physically mapped with the restriction endonucleases AvaI, AvaII, EcoRI, HpaI, KpnI, PvuII (one site each), HindIII, HaeII (three sites each), HincII (four sites), and HhaI (five sites). Using the cryptic plasmid pVA318 as cloning vehicle, the largest HindIII fragment of pSM10 (3.3 kb) was shown to contain the erythromycin/lincomycin resistance gene(s) of the plasmid. The AvaII site of pSM10 proved to be suitable as a site for cloning AvaII-generated chromosomal DNA fragments from a group C streptococcal strain in the Challis strain of Streptococcus sanguis (group) H. A detailed physical map of the chimeric plasmid pSM10221 (12.8 kb), a fusion product of pSM10 and the staphylococcal chloramphenicol resistance plasmid pC221 (4.5 kb), is also presented. The plasmid chimera has properties making it potentially useful in development of a doubly selective streptococcal cloning vehicle by searching for insertional inactivation.     

Glucocorticoid and retinoid regulation of alpha-2 type I procollagen promoter activity.

Glucocorticoids decrease type I procollagen synthesis by decreasing the steady state levels of procollagen mRNAs and mRNA synthesis. The present studies were undertaken to determine the functional sequences of the pro alpha 2(I) collagen gene required for the glucocorticoid-mediated decrease of type I procollagen mRNA synthesis. Embryonic mouse fibroblasts were stably transfected with the pR40 DNA CAT construct containing the 5' flanking region fragment from -2048 to +54 and the intronic fragment from +418 to +1524 of the mouse alpha 2(I) collagen gene. Dexamethasone treatment of these pR40 transfected fibroblasts resulted in a significant decrease in CAT activity which agrees with the glucocorticoid-mediated decrease of the steady state levels of type I procollagen mRNAs. To determine the possible role of the first intron fragment in the dexamethasone-mediated decrease of CAT activity, pR36, a CAT plasmid containing the first intron fragment and the SV40 early promoter, was transfected into mouse fibroblasts and treated with dexamethasone. No significant decrease in CAT activity was observed. The dexamethasone-mediated response was then localized within the 5' flanking region by preparing a series of constructs containing internal deletions and transfecting these plasmids into mouse fibroblasts. The regions -2048 to -981 and -506 to -351 were required for the dexamethasone response of gene activity. However, the DNA stretch from -981 to -506 was not. Analysis of the DNA sequences of these regions revealed a single GRE at -1023 to -1018 and a modified doublet at -873 to -856.(ABSTRACT TRUNCATED AT 250 WORDS)