A series of repetitive DNA sequences are associated with human core and H1 histone genes

Summary Repetitive DNA sequences, derived from the human β-globin gene cluster, were mapped within a series of human genomic DNA segments containing core (H2A, H2B, H3 and H4) and H1 histone genes. Cloned recombinant λCH4A phage with human histone gene inserts were analyzed by Southern blot analysis using the following³²P-labeled (nick translated) repetitive sequences as probes:Alu I,Kpn I and LTR-like. A cloned DNA designated RS002-5′C6 containing (i)a (TG)₁₆ simple repeat, (ii) an (ATTTT)n repeat and (iii)a 52 base pair alternating purine and pyrimidine sequence was also used as a radiolabelled hybridization probe. Analysis of 12 recombinant phage, containing 6 arrangements of core histone genes, indicated the presence ofAlu I,Kpn and RS002-5′C6 repetitive sequences. In contrast, analysis of 4 human genomic DNA segments, containing both core and H1 histone genes, indicated the presence of onlyAlu I family sequences. LTR-like sequences were not detected in association with any of the core or H1 histone genes examined. These results suggest that human histone and β-globin genes share certain aspects of sequence organization in flanking regions despite marked differences in their overall structure and pattern of expression.     

Analysis of CYP21A1P and the duplicated CYP21A2 genes

The RCCX module on chromosome 6p21.3 has 3 possible forms: monomodular, bimodular, and trimodular. Chromosomes with 4 RCCX modules are very rare. In the monomodule, most of the CYP21A1P genes do not exist. However, haplotypes of the RCCX module with more than one CYP21A2 gene were observed. Obviously, the gene located downstream of the XA gene can possibly include the CYP21A2 as well as the CYP21A1P gene.     

Immunofractionation of DNA sequences associated with HMG-17 in chromatin

Antibodies specific for chromosomal protein HMG-17 were immobilized on CNBr-Sepharose and the resulting immunoaffinity column was used to purify chromatin segments enriched in HMG-17. The DNA was purified from both the nucleosomal fraction which was bound to the column and from the fraction which was not bound, and examined with DNA probes representing repetitive DNA, non-transcribed genes, transcribed genes and inducible genes. The results suggest that HMG-17 is preferentially associated with DNA sequences coding for genes, regardless of whether they are transcribed, and therefore support the notion that HMG-17 confers specific structural characteristics on selected regions in the genome.     

Prostate cancer with variants in CYP17 and UGT2B17 genes: a meta-analysis

Both CYP17 and UGT2B17 are suggested to be potential risk factors of prostate cancer (PCa). To date, many studies have evaluated the relationship between CYP17 T-34C and UGT2B17 Del polymorphisms and Prostate cancer with conflicting results. Here, we performed comprehensive meta-analyses of over 25 studies, including results from about 17,000 subjects on the association of CYP17 T-34C and UGT2B17 Del polymorphisms with Prostate cancer. Overall, no significant associations between CYP17 T-34C polymorphism and Prostate cancer risk were found for T versus C (P=0.63), TT versus CC (P=0.52), TT+TC versus CC (P=0.40) or TT versus TC+CC (P=0.98), though there was a marginally significant association with the UGT2B17 Del polymorphism under Del/Del versus Ins/Ins +Ins/Del (P=0.05). In an analysis of various subgroups, there were no substantially significant associations with the CYP17 T-34C polymorphism; while there was a significant association for the UGT2B17 Del/Del genotype in a subgroup of men-based controls (P < 0.0001). The current meta-analysis results suggest that the CYP17 T-34C polymorphism may not be associated with Prostate cancer, while the UGT2B17 Del polymorphism may significantly contribute to prostate cancer susceptibility in men. These findings also support the idea that CYP17 has no significant effects on androgen levels, while UGT2B17 does.     

飞蝗CYP408B1和CYP409A1基因的原核表达

【目的】细胞色素P450是分布极其广泛的超家族酶,在昆虫内源及外源化合物代谢中发挥着重要的作用。本文分析了飞蝗Locusta migratoria CYP408B1和CYP409A1基因在不同组织部位的表达差异,并对两种蛋白进行原核表达,为其分子特性和生物学功能的深入研究提供基础资料。【方法】提取飞蝗5龄若虫不同组织部位的总RNA,体外反转录成c DNA,采用Real-time PCR和RT-PCR技术分析飞蝗CYP408B1和CYP409A1在不同组织部位的表达模式,构建表达载体p CW/CYP408B1、p CW/CYP409A1和p AC/CPR,将p CW/CYP408B1和p CW/CYP409A1分别与p AC/CPR在大肠杆菌Escherichia coli BL21(DE3)中进行共表达。【结果】通过PCR检测,发现CYP408B1和CYP409A1在飞蝗5龄若虫触角、脑、视叶、咽下神经节、胸神经节和附腺中均有表达,其中CYP408B1在附腺中表达量较高。原核表达结果显示,CYP409A1和CPR(NADPH细胞色素P450还原酶)均可表达,蛋白分子量分别约为58 ku和77 ku,但均为包涵体,而CYP408B1未能成功表达。【结论】本文揭示了飞蝗CYP408B1和CYP409A1在不同组织部位的表达模式,并对CYP409A1和CPR进行了原核表达,研究结果为深入探讨飞蝗细胞色素P450基因对杀虫剂的代谢解毒作用提供了实验依据和基础资料。     

Molecular cloning of sequences encoding the human heat-shock proteins and their expression during hyperthermia

Plasmids containing cDNA copies of mRNAs induced in HeLa cells by heat shock have been isolated and characterized. In vitro translation of RNAs selected by hybridization to plasmid DNAs identified sequences representing the three major classes (89, 70 and 27-kDa) of heat-shock proteins (hsp) and a 60-kDa minor hsp. Plasmids with inserts specific for the 27, 60, and 70-kDa hsp each hybridize with a single discrete size class of heat-inducible mRNA. Plasmids specific for the 89-kDa protein, however, hybridize with either a 2.7- or 2.95-kb mRNA species. Both mRNAs are coordinately induced during heat shock. We show that the characteristic pattern of induction and repression of each class of hsp during sustained hyperthermia is the result of changes in the steady state level of each mRNA.     

A cAMP-responsive element regulates expression of the mouse steroid 11 beta-hydroxylase gene

In Y1 mouse adrenocortical tumor cells, expression of steroid 11 beta-hydroxylase (11 beta-OHase) is stimulated by cAMP following a delay of 4-6 h. Our results demonstrate that a cAMP-responsive element (CRE) within the 11 beta-OHase promoter region is a major determinant of this induction. The 5'-flanking sequences from the mouse 11 beta-OHase gene were placed in front of a human growth hormone reporter gene and transfected into Y1 cells. Treatment of transfected cells with 8-bromo-cAMP increased expression directed by the 11 beta-OHase 5'-flanking region by 3.8-fold. In 5'-deletion analyses, 123 base pairs of 5'-flanking sequences were sufficient for cAMP induction, whereas cAMP treatment did not affect expression of a plasmid with only 40 base pairs of 5'-flanking sequence. Within these 123 base pairs, a region from -56 to -49 matched 7 of 8 bases comprising the consensus sequence for the CRE. 11 beta-OHase 5'-flanking sequences from -65 to -42, including the CRE-like sequence, conferred cAMP inducibility to promoters from the thymidine kinase and chorionic gonadotropin alpha-subunit genes. DNase I footprinting and Southwestern blotting analyses demonstrated that the protein which interacted with the CRE in the 11 beta-OHase promoter region was similar to the CRE-binding protein associated with other cAMP-regulated genes. Together, these results suggest that an interaction between the 11 beta-OHase CRE and CRE-binding protein mediates cAMP induction of the 11 beta-OHase gene.     

Localization of proteins, specifically binding highly-repetitive sequences of DNA in human spermatozoids

Immunoblot revealed in spermatozoa alpha-satellite (sat) DNA-specific centromere protein B (CENP-B) and p70 (Enukashvily et al., 2000), a membrane telomere binding protein (MTBP/TRF2) (Podgornaya et al., 2000), and Alu-binding protein p68 (Lukyanov et al., 2000). The localization of some of these proteins in spermatozoa was defined using indirect immunofluorescence. Spermatozoa were fixed in methanol/acetic acid 3:1, or prior to fixation were treated with 5 mM heparin and 10 mM DTT. The heparin/DTT treatment causes the nuclear membrane destruction and a partial chromatin decondensation. In non-treated spermatozoa fluorescent signals from all ABs are registered near the membrane, with MTBP/TRF2 being localized closer to the acrosome than sat-DNA-specific proteins. In the treated spermatozoa MTBP/TRF2 was partially lost, whereas part of CENP-B and sat-p70 remained in contact with membrane. Another part of sat-binding proteins reveals a dot-like staining pattern, with dots confined to the DAPI-stained chromatin area, inside a nuclei. This is in partial agreement with the pattern of telomere and CEN position revealed by FISH. Commonly MTBP has a near membrane localization, being lost when the nuclear membrane is destroyed. Centromere-binding proteins are arranged in the order from the nuclear membrane towards the nuclear center, with CENP-B being situated more peripherally but not in the middle of the nucleus. This discrepancy may be explained by the fact, that some proteins are not associated with the appropriate sequences in a spermatozoon. Possibly, such a distribution of proteins may reflect their role in unpacking the paternal genetic material in a zygote.     

Homology modeling and substrate binding study of human CYP4A11 enzyme

Although both bacterial CYP102 (P450BM3) and mammalian CYP4A isozymes share a common function as fatty acid hydroxylases, distinctly different preferred sites of oxidation are observed with the CYP102 performing the usual non-terminal hydroxylation or epoxidation and the CYP4A enzymes performing the unusual and enigmatic terminal hydroxylation. The origin of this unique product specificity in human CYP4A11 has been explored in this work, focusing on possible differences in the binding site architecture of the two isozymes as the cause. To this end, 3D model structures of the human CYP4A11 enzyme were built and compared to the X-ray structure of CYP102. The substrate-binding channel identified in CYP4A11 was found to have a much more sterically restricted active site than that in CYP102 that could cause limited access of long-chain fatty acid to the ferryl oxygen leading to the preferred omega-hydroxylation. Results of docking of a common substrate, lauric acid, into the binding site of both CYP4A11 and CYP102 and molecular dynamics simulations provided additional support for this hypothesis. Specifically, in the CYP4A11-lauric acid simulations, the omega hydrogens were closest to the ferryl oxygen most of the time. By contrast, in the CYP102-lauric acid complex, the substrate could penetrate further into the active site providing access of the non-terminal (omega-1, omega-2) positions to the ferryl oxygen. These results, taken together, have elucidated the origin of the unusual product specificity of CYP4A11 and illustrated the central role of binding site architecture in subtle modulation of function.     

Exploring QSAR for CYP11B2 binding affinity and CYP11B2/CYP11B1 selectivity of diverse functional compounds using GFA and G/PLS techniques

A data set of a series of 132 structurally diverse compounds with cytochrome 11B2 and 11B1 (CYP11B2 and CYP11B1) enzyme inhibitory activities was subjected to molecular shape analysis to explore contributions of shape features as well as electronic, structural, and physicochemical parameters toward enzyme inhibitory activities, in search of appropriate molecular scaffolds with optimum substitutions for highly potent CYP11B2 inhibitors. Genetic function approximation (GFA) and genetic partial least squares (G/PLS) were used as chemometric tools for modeling, and the derived equations were of acceptable statistical quality considering both internal and external validation parameters (Q²: 0.514–0.659, R²_pred: 0.510–0.734). The G/PLS models with spline option for CYP11B2 and CYP11B1 inhibition and selectivity modeling appeared to be the best models based on r_m²_(overall) criterion. The study indicates the importance of the pyridinylnaphthalene and pyridylmethylene-indane scaffolds with less polar and electrophilic substituents for optimum CYP11B2 inhibitory activity and CYP11B2/CYP11B1 selectivity.     

Analysis of the CYP21A1P pseudogene: indication of mutational diversity and CYP21A2-like and duplicated CYP21A2 genes

The CYP21A1P gene downstream of the XA gene, carrying 15 deteriorated mutations, is a nonfunctional pseudogene that shares 98% nucleotide sequence homology with CYP21A2 located on chromosome 6p21.3. However, these mutations in the CYP21A1P gene are not totally involved in each individual. From our analysis of 100 healthy ethnic Chinese (i.e., Taiwanese) (n = 200 chromosomes) using the polymerase chain reaction (PCR) products combined with an amplification-created restriction site (ACRS) method and DNA sequencing, we found that approximately 10% of CYP21A1P alleles (n = 195 chromosomes) presented the CYP21A2 sequence; frequencies of P30, V281, Q318, and R356 in that locus were approximately 24%, 21%, 11%, and 34%, respectively, and approximately 90% of the CYP21A1P alleles had 15 mutated loci. In addition, approximately 2.5% (n = 5 chromosomes) showed four haplotypes of the 3.7-kb TaqI-produced fragment of the CYP21A2-like gene and one duplicated CYP21A2 gene. We conclude that the pseudogene of the CYP21A1P mutation presents diverse variants. Moreover, the existence of the CYP21A2-like gene is more abundant than that of the duplicated CYP21A2 gene downstream of the XA gene and could not be distinguished from the CYP21A2–TNXB gene; thus, it may be misdiagnosed by previously established methods for congenital adrenal hyperplasia caused by a 21-hydroxylase deficiency.     

Identification and expression analysis of genes associated with bovine blastocyst formation

Background  

Normal preimplantation embryo development encompasses a series of events including first cleavage division, activation of the embryonic genome, compaction and blastocyst formation.     

A short repeat in the genome of the DNA sequences flanking bovine growth hormone genes

The phage clones containing a gene coding for bovine growth hormone were isolated from a bovine genomic library. Comparison of the 5' and 3' regions flanking the bovine growth hormone gene by Southern blot hybridization revealed that they share homology. Screening the bovine genomic library by nick-translated DNA fragment from 5' flanking region leads to conclusion that this sequence is present in 0.1% of clones. Each analysed clone carrying the sequence contains some copies of it.