Isolation of the chicken middle-molecular weight neurofilament (NF-M) gene and characterization of its promoter.

We have isolated and sequenced genomic DNA clones covering the coding region of the chicken mid-size neurofilament (NF-M) gene and greater than 1 kb of its 5' upstream region. The NF-M gene contains two introns which both are located within the highly conserved C-terminal region of the rod domain. The 5' end of the corresponding mRNA was assigned to a G residue 40 nucleotides upstream of the translation start site and in appropriate distance from a potential TATA box. To functionally analyze the NF-M promoter, constructs carrying 112, 222, and 1026 nucleotides of the 5' upstream region in front of a luciferase reporter gene were tested for their capability to direct luciferase expression after transient transfection into various cell lines. Significant luciferase activity was recorded both in rat phaeochromocytoma (PC12) cells and murine fibroblasts. In PC12 cells, in which neurite outgrowth is induced by nerve growth factor (NGF), expression was stimulated up to 13-fold within 3 days of NGF treatment. This closely resembles expression of the endogenous NF-M gene in response to this hormone.     

Characterization of two mRNA species encoding human estradiol 17 beta-dehydrogenase and assignment of the gene to chromosome 17

Using two 33-mer synthetic oligonucleotides derived from the amino acid sequence of the catalytic site of estradiol 17 beta-dehydrogenase (E2DH) and polyclonal antibodies raised against the enzyme purified from human placenta, clones were isolated from a lambda gt11 human placental cDNA library. A 327-amino acid sequence was deduced from cDNA sequencing. Two mRNA species have been identified in poly(A)+ RNA from human placenta, a major species migrating at 1.3 kb while a minor one is found at approx. 2.2 kb. Primer extension and S1 nuclease analysis indicate that the major mRNA species starts 9-10 nucleotides while the minor mRNA starts 971 nucleotides upstream from the ATG initiating codon, respectively. Sequence analysis of the longest cDNA clone (2092 bp) shows that it possesses identical coding and non-coding sequences in the regions of overlap with the shorter cDNA clones. The 32P-labeled 5' non-coding fragment hybridizes only to the 2.2 kb band, thus providing evidence for the existence of two distinct mRNA species which differ in their 5' noncoding regions. Using hp E2DH-36 cDNA as a probe for in situ hybridization of translocated chromosomes, the human E2DH gene was localized to the q11-q12 region of chromosome 17.     

Glutathione transferase pi its minimal promoter and downstream cis-acting element.

Fragments of the human glutathione S-transferase pi gene and 15 kb of its 5' flanking region have been fused to the chloramphenicol acetyl transferase (CAT) reporter gene. Transfection into a number of human cell lines (Hela, HepG2, MCF7 and EJ) has demonstrated that the AP1 binding site, located between nucleotides -58 and -65 (Cowell et al. 1988. Biochem. J. 255, 79-83), is essential for basal level promoter activity. We have also identified a positive cis-acting DNA element between nucleotides +8 and +72 which seems to be part of the promoter. No other regulatory activity was identified within the 17 kb analyzed.     

Molecular cloning and characterization of the complementary DNA and gene coding for the B-chain of subcomponent C1q of the human complement system.

Plasmid clones containing cDNA coding for the B-chain of human Clq were isolated from a liver cDNA library. The longest cDNA insert isolated contained all the coding sequence for amino acid residues B1 to B226 plus a 3' non-translated region of 264 nucleotides that extended into the poly(A) tail, thus accounting for 950 nucleotides of the mRNA. The B-chain mRNA was estimated by Northern-blot analysis to be 1.46 kb (kilobases) long, which indicated that approx. 500 bases were not accounted for in the cDNA clone. A cosmid clone containing the C1q-B chain gene was isolated from a human genomic DNA library. The precise 5' limit of gene was not established, but from the data available it appears that the gene is approx. 2.6 kb long. The coding sequence for residues B1 to B226 in the gene is interrupted by one intron, of 1.1 kb, which is located within the codon coding for glycine at position B36. This glycine residue is located in the middle of the triple-helical regions found in C1q at exactly the position where there is an unusual structural feature, i.e. a bend in each of the helical regions brought about by the interruption of the Gly-Xaa-Yaa repeating triplet sequences in the A- and C-chains and the presence of an 'extra' triplet in the B-chain. Nucleotide sequencing of the 5' end of the gene indicates the presence of a predominantly hydrophobic stretch of 29 amino acids, immediately before residue B1, which could serve as a signal peptide.     

Discrimination among multiple AATAAA sequences correlates with interspecies conservation of select 3'' untranslated nucleotides.

The DNA sequence corresponding to the 1.3 kb 3' untranslated region of the 6.5 kb human procollagen alpha 1(IV) mRNA was determined and compared with the mouse sequence obtained from 3' cDNA and genomic clones overlapping the reported 5' half (Oberbaumer et al., 1985, Eur. J. Biochem. 147:217). Although four AAUAAA hexanucleotides are found in the human and seven in the mouse RNAs, Northern blot hybridization showed almost exclusive utilization of the most 3' sequence, in contrast to the pattern seen when using alpha 1(I), alpha 2(I), alpha 1(III) and alpha 2(V) procollagen probes. Moreover, the ninety nucleotides 5' to the poly A tail in the major alpha 1(IV) mRNAs exhibit a much greater degree of interspecies homology than those encompassing the other three shared AAUAAA recognition signals. Further examination of this highly conserved area revealed the presence of two "consensus sequences" found in the 3' noncoding region of a number of RNA polymerase II transcribed genes (Mattaj and Zeller, 1983, Embo J. 2:1883) and, unexpectedly, some similarity with the nucleotides 5' to the poly A attachment signals in other procollagen mRNAs.     

人同源异型盒基因Hu-1位点的RFLPs研究

用12种限制性核酸内切酶分别对4至104条人类染色体进行酶谱分析,以寻找Hu-1基因附近的限制性酶切多态位点,仅发现了Bg 1Ⅱ酶的一个多态性位点。表现为Bg 1Ⅱ酶谱中除3.6kb片段外,杂合子型尚具6.3及6.6kb两种片段,而纯合子型则只有6.3或6.6kb一种片段。在所检查的104条染色体中6.6kb片段出现的频率为3.88％,而6.3kb片段出现的频率为96.12％。经统计分析,计算出Hu-1基因及其附近的核苷酸变异率为0.0017,大体上与人β珠蛋白基因簇及人α-1-抗胰蛋白酶基因的核苷酸变异率相似。文中对本工作的意义进行了初步讨论。     

Molecular cloning, expression and chromosomal localization of a novel human REG family gene, REG III

Regenerating gene (Reg), first isolated from a regenerating islet cDNA library, encodes a secretory protein with a growth stimulating effect on pancreatic beta cells that ameliorates the diabetes of 90% depancreatized rats and non-obese diabetic mice. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of four subtypes (types I, II, III, IV) based on the primary structures of the encoded proteins of the genes [Diabetes 51(Suppl. 3) (2002) S462]. Plural type III Reg genes were found in mouse and rat. On the other hand, only one type III REG gene, HIP/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitis-associated protein), was found in human. In the present study, we found a novel human type III REG gene, REG III. This gene is divided into six exons spanning about 3 kilobase pairs (kb), and encodes a 175 amino acid (aa) protein with 85% homology with HIP/PAP. REG III was expressed predominantly in pancreas and testis, but not in small intestine, whereas HIP/PAP was expressed strongly in pancreas and small intestine. IL-6 responsive elements existed in the 5'-upstream region of the human REG III gene indicating that the human REG III gene might be induced during acute pancreatitis. All the human REG family genes identified so far (REG Ialpha, REG Ibeta, HIP/PAP, REG III and REG IV) have a common gene structure with 6 exons and 5 introns, and encode homologous 158-175-aa secretory proteins. By database searching and PCR analysis using a yeast artificial chromosome clone, the human REG family genes on chromosome 2, except for REG IV on chromosome 1, were mapped to a contiguous 140 kb region of the human chromosome 2p12. The gene order from centromere to telomere was 5' HIP/PAP 3'-5' RS 3'-3' REG Ialpha 5'-5' REG Ibeta 3'-3' REG III 5'. These results suggest that the human REG gene family is constituted from an ancestor gene by gene duplication and forms a gene cluster on the region.