The E6 and E7 genes of human papillomavirus type 6 have weak immortalizing activity in human epithelial cells.

Previous studies have shown that the E7 gene of human papillomavirus (HPV) type 16 or 18 alone was sufficient for immortalization of human foreskin epithelial cells (HFE) and that the efficiency was increased in cooperation with the respective E6 gene, whereas the HPV6 E6 or E7 gene was not active in HFE. To detect weak immortalizing activities of the HPV6 genes, cells were infected with recombinant retroviruses containing HPV genes, alone and in homologous and heterologous combinations. The HPV6 genes, alone or together (HPV6 E6 plus HPV6 E7), were not able to immortalize cells. However the HPV6 E6 gene, in concert with HPV16 E7, increased the frequency of immortalization threefold over that obtained with HPV16 E7 alone. Interestingly, 6 of 20 clones containing the HPV16 E6 gene and the HPV6 E7 gene were immortalized, whereas neither gene alone was sufficient. Thus, the HPV6 E6 and E7 genes have weak immortalizing activities which can be detected in cooperation with the more active transforming genes of HPV16. Acute expression of the HPV6 and HPV16 E6 and E7 genes revealed that only HPV16 E7 was able to stimulate the proliferation of cells in organotypic culture, resulting in increased expression of the proliferative cell nuclear antigen and the formation of a disorganized epithelial layer. Additionally, combinations of genes that immortalized HFE cells (HPV16 E6 plus HPV16 E7, HPV16 E6 plus HPV6 E7, and HPV6 E6 plus HPV16 E7) also stimulated proliferation.     

High homology between 6-aminohexanoate-cyclic-dimer hydrolases of Flavobacterium and Pseudomonas strains.

The nucleotide sequences of the genes for 6-aminohexanoate-cyclic-dimer hydrolases of Flavobacterium sp. strain K172 (F-nylA) and Pseudomonas sp. NK87 (P-nylA), enzymes essential for the degradation of a by-product of the nylon-6 industry, were obtained by the dideoxynucleotide chain-termination method. A 1,479-base-pair open reading frame starting at a GTG and terminating at a TGA was found for the both of the genes. The P-nylA and F-nylA genes encoded polypeptides of 493 amino acids and had only 10 base substitutions in the coding region, which caused seven amino acid substitutions.     

Chicken interleukin-6. cDNA structure and biological properties.

Suppression subtractive hybridization technology was used to identify differentially expressed genes in spleens of chickens that had been treated with the synthetic immune modifier S-28463. One induced chicken gene encoded a protein with about 35% sequence identity to human interleukin-6 (IL-6). It consists of 241 amino acids including a putative N-terminal signal peptide of 47 residues. Bacterially expressed chicken IL-6 (ChIL-6) carrying a histidine tag in place of the signal peptide was biologically active: it induced proliferation of the IL-6-dependent murine hybridoma cell line 7TD1. The concentration of ChIL-6 required for half-maximal proliferative response was approximately 60 pg.mL-1. When injected intravenously into adult chickens, purified recombinant ChIL-6 induced an increase in serum corticosterone levels. Supernatants of chicken LMH and monkey COS-7 cells transiently transfected with a ChIL-6 expression construct induced proliferation of 7TD1 cells, demonstrating that recombinant ChIL-6 from eukaryotic cells is also active.     

Relationship between bacteriophage T4 and T6 DNA topoisomerases. T6 39-protein subunit is equivalent to the combined T4 39- and 60-protein subunits

T6 DNA topoisomerase has been purified from bacteriophage T6 infected Escherichia coli. Unlike the T4 DNA topoisomerase which has three subunits, it consists of two subunits of molecular weights 75,000 and 51,000. They are the products of T6 genes 39 and 52, respectively. The purified T6 enzyme can stimulate in vitro T6 DNA replication. It has an ATP-dependent DNA relaxation activity similar to the T4 enzyme. Either ATP or dATP can be used in both reactions. Using a "Western blotting" and radioimmuno-detection methods, we show that T6 39 subunit contains protein sequences specified by both the T4 39 and 60 genes. The 52-proteins of both phages appear to be identical. The T4 and T6 topoisomerase genes represent a naturally occurring example of gene separation or fusion.     

TCDD-up-regulation of IGFBP-6 and IL-5R alpha subunit genes in vivo and in vitro.

Although the potent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been well known for its immunosuppressive activity, the mechanisms of its action have been difficult to elucidate. This is partly due to its inability to exert its effects in vitro. To further elucidate the underlying mechanisms of TCDD effects, we screened for genes that are regulated by the in vivo TCDD treatment of mice that are challenged with allogeneic tumor cells. RNA, collected from lymphoid organs including the thymus, draining lymph nodes, and bone marrow, was reverse-transcribed to cDNA and hybridized to DNA arrays that consisted of 588 genes (ClonTech, USA). The expression of the NF-kappaB p65, c-jun, and p27(Kip1) genes was increased by the TCDD treatment, as previously reported. In addition, we found that the expression of several genes, which were not reported as regulated by TCDD, were modulated by TCDD. Some genes, including insulin-like growth factor-binding protein-6 (IGFBP-6) and IL-5R alpha, were upregulated; while other genes, including CD14, were down-regulated. The expression of the IGFBP-6 and IL-5R alpha subunit genes by TCDD in the thymus was confirmed by RT-PCR and Western blot analyses. Furthermore, TCDD effects on the expression of the IGFBP-6 gene was also observed with EL4 mouse thymoma cells. This suggests that IGFBP-6 may be involved in thymic atrophy, and EL4 cells may be used as an in vitro model for studying molecular mechanisms of thymic atrophy.     

Localization of two human homologs, HHR6A and HHR6B, of the yeast DNA repair gene RAD6 to chromosomes Xq24-q25 and 5q23-q31.

The chromosomal localizations of two closely related human DNA repair genes, HHR6A and HHR6B, were determined by in situ hybridization with biotinylated probes. HHR6A and HHR6B (human homolog of yeast RAD6) encode ubiquitin-conjugating enzymes (E2 enzymes), likely to be involved in postreplication repair and induced mutagenesis. The HHR6B gene was assigned to human chromosome 5q23-q31, whereas the HHR6A gene was localized on the human X chromosome (Xq24-q25). This latter assignment was confirmed with an X-specific human-mouse/hamster somatic cell hybrid panel. Southern blot analysis points to an X and an autosomal localization of HHR6A and HHR6B, respectively, in the mouse. The potential involvement of these genes in human genetic disorders is discussed.     

Pax 6: mastering eye morphogenesis and eye evolution.

Pax 6 genes from various animal phyla are capable of inducing ectopic eye development, indicating that Pax 6 is a master control gene for eye morphogenesis. It is proposed that the various eye-types found in metazoa are derived from a common prototype, monophyletically, by a mechanism called intercalary evolution.     

Identification of psiB genes of plasmids F and R6-5. Molecular basis for psiB enhanced expression in plasmid R6-5.

PsiB protein of plasmid R6-5 inhibits the induction of the SOS pathway. The F sex factor also carries a psiB gene homologous to that of R6-5. Yet, it fails to inhibit SOS induction. In order to solve this difference, we characterized the psiB genes of R6-5 and F. We found that (i) the sequences of the two psiB genes share extensive homology the predicted amino acid sequences of the two proteins differing by 5 residues, (ii) the expression of R6-5 psiB is 4 times higher than F psiB gene, (iii) in plasmid R6-5, a Tn10 transposon upstream from the psiB gene enhances psiB expression. Hence, the F sex factor may be unable to prevent SOS induction for two non-exclusive reasons: (i) F PsiB protein, being slightly different from R6-5, may be less active, (ii) the level of synthesis of F PsiB protein may be insufficient to prevent SOS induction.     

Genetic variation in RPS6KA1, RPS6KA2, RPS6KB1, RPS6KB2, and PDK1 and risk of colon or rectal cancer

RPS6KA1, RPS6KA2, RPS6KB1, RPS6KB2, and PDK1 are involved in several pathways central to the carcinogenic process, including regulation of cell growth, insulin, and inflammation. We evaluated genetic variation in their candidate genes to obtain a better understanding of their association with colon and rectal cancer. We used data from two population-based case-control studies of colon (n=1574 cases, 1940 controls) and rectal (n=791 cases, 999 controls) cancer. We observed genetic variation in RPS6KA1, RPS6KA2, and PRS6KB2 were associated with risk of developing colon cancer while only genetic variation in RPS6KA2 was associated with altering risk of rectal cancer. These genes also interacted significantly with other genes operating in similar mechanisms, including Akt1, FRAP1, NFκB1, and PIK3CA. Assessment of tumor markers indicated that these genes and this pathway may importantly contributed to CIMP+ tumors and tumors with KRAS2 mutations. Our findings implicate these candidate genes in the etiology of colon and rectal cancer and provide information on how these genes operate with other genes in the pathway. Our data further suggest that this pathway may lead to CIMP+ and KRAS2-mutated tumors.     

表达毒素源性大肠杆菌定居因子抗原CS6的一组基因的克隆

人源毒素源性大肠杆菌(Enterotoxigenic E.Coil ETEC)是引起婴幼儿及旅游者腹泻的主要致病菌。ETEC的致病因子包括定居因子抗原 (Colonization factor antigens,CFAs)和肠毒素。大多数ETEC菌株均能产生抗原特异的菌毛,介导细菌与宿主小肠粘膜表面并在粘膜表面上受体的结合,使得细菌能定殖于粘膜表面并在粘膜的功能性清除中存活下来。已经报道的定居因子抗原包括CFA/Ⅰ,CFA/Ⅱ、CFA/Ⅲ、CFA/Ⅳ(PCF8775)、PCF09和PCFO159等。CFA/Ⅰ和CFA/Ⅲ均由单一的菌毛亚基构成,CFA/Ⅱ、CFA/Ⅳ则由多种表面抗原(colisurface antigen,CS)复合而成。所有的CFA/Ⅳ阳性菌株均表达CS6抗原,其中有些同时表达CS4或CS5抗原。CS4和CS5都是直径6～7nm的菌毛,能引起人和牛血红细胞的甘露糖抗性的凝集反应(MRHA)。迄今为止,没有观祭到CS6明显的菌毛结构,CS6也不能引起MRHA阳性反应。然而动物实验的结果表明,CS6是CFA/Ⅳ复合抗原中一种对定居作用最重要的抗原”,。本研究组已经通过分子克隆的手段获得了能分别表达CFA/Ⅰ和CS3抗原的重组菌株,用于人源ETEC疫苗的研究。本文报道了表达CS6菌毛抗原的DNA片段的克隆,并用免疫吸附制备的CS6抗血清测定了重组菌株表达的菌毛蛋白。     

Characterization of Gordonia sp. strain CC-NAPH129-6 capable of naphthalene degradation

A naphthalene-degrading isolate able to utilize naphthalene as a sole carbon source was identified as Gordonia sp. CC-NAPH129-6. Here a detail characterization of the naphthalene catabolic genes present in this strain was conducted. In nar region four structural genes (narAa, narAb, narB, narC), two regulatory genes (narR1, narR2), a rubredoxin encoding gene (rub1) and a gene (orf7) with unknown function were obtained. When compared with most of the members within naphthalene-degrading Rhodococcus, these naphthalene catabolic genes in strain CC-NAPH129-6 were organized into an operon-like gene cluster and present in the same order. This naphthalene gene cluster located in a 97-kb small plasmid of strain CC-NAPH129-6, as can be seen from the PFGE and Southern blot hybridization data. Besides, a partial transposase sequence containing an IS element structure with 12-nt inverted repeat at both ends was found, which was flanked by direct repeats downstream the narC gene in strain CC-NAPH129-6. This novel transposase gene sequence was unlike to the transposase sequence found between narR2 and rub1 genes in Rhodococcus opacus R7. The comparative analyses of the naphthalene catabolic genes, 16S rRNA and gyrB gene present in strain CC-NAPH129-6 and naphthalene-degrading Rhodococcus species imply that the naphthalene catabolic genes in strain CC-NAPH129-6 might be horizontally transferred from Rhodococcus members. This is the first report demonstrating that naphthalene catabolic genes organized into an operon-like gene cluster in the genus Gordonia, and this might provide evidence of the importance of this actinobacterial lineage in the bioremediation of oil-contaminated soils.     

Discovery of a novel murine keratin 6 (K6) isoform explains the absence of hair and nail defects in mice deficient for K6a and K6b

The murine genome is known to have two keratin 6 (K6) genes, mouse K6 (MK6)a and MK6b. These genes display a complex expression pattern with constitutive expression in the epithelia of oral mucosa, hair follicles, and nail beds. We generated mice deficient for both genes through embryonic stem cell technology. The majority of MK6a/b-/- mice die of starvation within the first two weeks of life. This is due to a localized disintegration of the dorsal tongue epithelium, which results in the build up of a plaque of cell debris that severely impairs feeding. However, approximately 25% of MK6a/b-/- mice survive to adulthood. Remarkably, the surviving MK6a/b-/- mice have normal hair and nails. To our surprise, we discovered MK6 staining both in the hair follicle and the nail bed of MK6a/b-/- mice, indicating the presence of a third MK6 gene. We cloned this previously unknown murine keratin gene and found it to be highly homologous to human K6hf, which is expressed in hair follicles. We therefore termed this gene MK6 hair follicle (MK6hf). The presence of MK6hf in the MK6a/b-/- follicles and nails offers an explanation for the absence of hair and nail defects in MK6a/b-/- animals.