Effects of UV, 4-NQO and TPA on gene expression in cultured human epidermal keratinocytes.

In an approach to study effects of UV light on gene expression in human epidermal keratinocytes, a cDNA library was constructed from poly(A)RNA isolated after UV irradiation from cultured keratinocytes. The cDNA library was differentially screened with labelled cDNA probes synthesized on poly(A)RNA isolated from UV irradiated or nonirradiated keratinocytes. Forty clones were selected and subjected to further analysis, 31 of them are described in this report. Whereas total mRNA synthesis is reduced after UV irradiation or treatment with 4-NQO Northern blot analysis revealed that there is an at least relative increase in the level of mRNAs corresponding to the majority of the isolated cDNA clones. Among these 15 were identified as corresponding to mRNAs for 50K and 56K keratins and for 50K- and 46K-related keratin. In addition, clones were found corresponding to the proteinase inhibitor cystatin A and to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Treatment of keratinocytes with the tumor promoter TPA had no effect on the mRNA level for most of the clones except those corresponding to keratins. Our results indicate that in keratinocytes UV irradiation leads to a relative increase in the level of some mRNAs.     

Molecular cloning and biological characterization of the human excision repair gene ERCC-3.

In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent mutants. After selection of UV-resistant primary and secondary 27-1 transformants, human sequences associated with the induced UV resistance were rescued in cosmids from the DNA of a secondary transformant by using a linked dominant marker copy and human repetitive DNA as probes. From coinheritance analysis of the ERCC-3 region in independent transformants, we deduce that the gene has a size of 35 to 45 kilobases, of which one essential segment has so far been refractory to cloning. Conserved unique human sequences hybridizing to a 3.0-kilobase mRNA were used to isolate apparently full-length cDNA clones. Upon transfection to 27-1 cells, the ERCC-3 cDNA, inserted in a mammalian expression vector, induced specific and (virtually) complete correction of the UV sensitivity and unscheduled DNA synthesis of mutants of complementation group 3 with very high efficiency. Mutant 27-1 is, unlike other mutants of complementation group 3, also very sensitive toward small alkylating agents. This unique property of the mutant is not corrected by introduction of the ERCC-3 cDNA, indicating that it may be caused by an independent second mutation in another repair function. By hybridization to DNA of a human x rodent hybrid cell panel, the ERCC-3 gene was assigned to chromosome 2, in agreement with data based on cell fusion (L. H. Thompson, A. V. Carrano, K. Sato, E. P. Salazar, B. F. White, S. A. Stewart, J. L. Minkler, and M. J. Siciliano, Somat. Cell. Mol. Genet. 13:539-551, 1987).     

Human plasma inter-alpha-trypsin inhibitor is encoded by four genes on three chromosomes

Human inter-alpha-trypsin inhibitor is a plasma protein of Mr 180,000 which has long been described as a single polypeptide chain. However, we have previously demonstrated that it is synthesized in liver by two different mRNA populations coding for heavy or light polypeptide chains [Bourguignon, J. et al. (1983) FEBS Lett. 162, 379-383] and cDNA clones for the heavy or light chains have recently been isolated and characterized [Bourguignon, J. et. al. (1985) Biochem. Biophys. Res. Commun. 131, 1146-1153; Salier, J.P. et al. (1987) Proc. Natl Acad. Sci. USA 84, 8272-8276]. In the present study, we show that human poly(A)-rich RNAs hybrid-selected with various heavy-chain-encoding cDNA clones translate three different heavy chains, designated H1 (Mr 92,000), H2 (Mr 98,000) and H3 (Mr 107,000). We previously characterized two heavy-chain cDNA clones. We now report that they correspond to H1 and H2 chains. We have also determined the sequence of an additional cDNA clone which codes for H3 chain. Its insert size is 1.79 kb with a single open reading frame and a poly(A) tail. The deduced amino acid sequence of the H3 chain is highly similar to those of the H1 (54%) and H2 (44%) chains. Northern analysis of human liver poly(A)-rich RNAs with the three heavy-chain cDNAs as probes clearly identified a single major mRNA population of 3.3 +/- 0.1 kb. Chromosomal localization by in situ hybridization shows that inter-alpha-trypsin inhibitor genes are located on three different human chromosomes. The H1 and H3 genes are located in the p211-p212 region of chromosome 3, whereas the H2 gene resides in the p15 band of chromosome 10. The light-chain gene is located in the q32-q33 region of chromosome 9. These results indicate that heavy and light chains of inter-alpha-trypsin inhibitor are encoded by at least four functional genes.     

Genomic structure of the human mitochondrial aldehyde dehydrogenase gene

We have isolated and characterized four overlapping clones from two cosmid human genomic libraries, which span about 90 kilobase pairs (kbp) and contain the entire human mitochondrial aldehyde dehydrogenase (ALDH2) gene. Restriction maps of the genomic clones were elucidated utilizing cDNA probes and specific oligonucleotide probes. The organization of exons and introns was established by DNA sequencing of each exon and splicing junctions. The ALDH2 gene is about 44 kbp in length and contains at least 13 exons which encode 517 amino acid residues. Except for the signal NH2-terminal peptide, which is absent in the mature enzyme, the amino acid sequence deduced from the exons coincided with the reported primary structure of human liver ALDH2 (J. Hempel, R. Kaiser, and H. J?rnvall, 1985, Eur. J. Biochem. 153: 13-28). Several introns contain Alu repetitive sequences. A TATA-like sequence (TTATAAAA) and a CAAT-like sequence (GTCATCAT) are located 473 and 515 bp, respectively, upstream from the translation initiation codon. Primer extension and S1 nuclease mapping were performed to characterize the 5'-region of the gene.     

Cloning of eukaryotic genes in single-strand phage vectors: the human interferon genes

Using oligonucleotide probes with defined sequences, we have selected clones from a human lymphocyte cDNA library which represent human leukocyte (HuIFN-α) and fibroblast (HuIFN-β) interferon gene sequences. Double-stranded f1 phage DNA was used as the vector for initial cloning of cDNA. Clones carrying interferon gene sequences were identified by hybridization with the oligonucleotide probes. The same oligonucleotide probes were used as primers for dideoxy chain termination sequencing of the clones. One HuIFN-α clone, 201, has a nucleotide sequence different from published HuIFN-α sequences. Under control of the lacUV5 promoter, the 201 gene has been used to express biologically active HuIFN-α in Escherichia coli.     

Molecular cloning and chromosomal localization of a human peripheral-type benzodiazepine receptor.

The sequencing of endopeptidase-generated peptides from the peripheral binding site (PBS) for benzodiazepines, purified from a Chinese hamster ovary (CHO) cell line, produced internal sequence information, and confirmed and extended the NH2-terminal PBS sequence that we previously reported. Since the sequences were highly similar to the corresponding rat PBS sequences, we investigated whether they were also conserved in human PBS. Scatchard analysis of [3H]PK11195 (a derivative of isoquinoline carboxamide) binding and photoaffinity labeling with [3H]PK14105 (a nitrophenyl derivative of PK11195) revealed that CHO PBS and human PBS are closely related. Furthermore a rabbit antiserum raised against three peptides synthesized on the basis of the CHO PBS sequence immunoprecipitate the solubilized U937 PBS and also recognize the human protein in an immunoblot analysis. Based on these results, we screened a U937 cell cDNA library with four oligonucleotide probes derived from the CHO sequence. Two of the probes hybridized with several clones that we isolated and sequenced. One of these, h-pPBS11, is 831 nucleotides and contains a full-length representation of human PBS mRNA. The amino acid sequence of human PBS deduced from the cDNA is 79% identical to that reported for rat PBS, however, human PBS contains two cysteines while rat PBS is characterized by the absence of this amino acid. Using the cDNA of human PBS as a probe, the PBS gene was located in the 22q13.3 band of the human genome.     

Cloning of the cDNAs encoding the cellular retinaldehyde-binding protein from bovine and human retina and comparison of the protein structures

A 1173-base pair cDNA encoding bovine cellular retinaldehyde-binding protein (CRALBP) was cloned from a bovine retinal cDNA expression library using as probes both anti-CRALBP polyclonal and monoclonal antibodies. The amino acid sequence deduced from the cDNA corresponds exactly to that determined by direct analysis of NH2-terminally acetylated bovine CRALBP (Crabb, J. W., Johnson, C. M., Carr, S. A., Armes, L. G., and Saari, J. C. (1988) J. Biol. Chem. 263, 18678-18687). Nick-translated bovine CRALBP cDNA probes were then used to clone from a human retinal cDNA library a 1317-base pair cDNA encoding human CRALBP. Bovine and human CRALBP are 92% identical in amino acid sequence and not related to any other known protein sequence. Both the bovine and human proteins contain 316 residues and have calculated molecular weights of 36,378 and 36,347, respectively, exclusive of the NH2-terminal blocking groups. The CRALBP cDNA clones should prove valuable as tools for studying the physiological role of the protein in vision and visual disorders.     

Molecular cloning of cDNA corresponding to mRNA species whose steady state levels in the thyroid are enhanced by thyrotropin. Homology of one of these sequences with ferritin H

A lambda gt11 cDNA library was constructed using poly(A)+ mRNA from thyrotropin (TSH)-stimulated Fisher rat thyroid (FRTL5) cells. The library was screened for nonthyroglobulin cDNA sequences by differential plaque filter hybridization using single-stranded cDNA probes synthesized from mRNA prepared from quiescent and TSH-stimulated FRTL5 cells. Thyroglobulin cDNA-containing recombinants in the library were avoided by prehybridizing the TSH probe to excess thyroglobulin cDNA. Of 48,000 clones screened, 60 were chosen as representing mRNA species whose abundance was increased in TSH-stimulated versus quiescent cultures. Southern blot analysis of 9 clones confirmed that the TSH-cDNA probe hybridized to a greater extent to the cDNA inserts than did the control probe. cDNA insert sizes varied between 0.3 kilobase (kb) and 1.0 kb. Northern slot blot analysis using as probes the cDNA of four of these clones (FC4, FC26, FC29, and FC43) demonstrated that TSH stimulation of FRTL5 cells increased the steady state levels of the respective mRNA species by 4-12-fold. For all 4 clones, increases in mRNA levels were apparent within approximately 1 h and were maximal after 14-18 h of TSH stimulation. Determination of the partial nucleotide sequence of these 4 clones confirmed that none was thyroglobulin, thyroid peroxidase, or any other gene previously reported to be stimulated by TSH. Three of the clones bore no homology to any known nucleotide sequence, but FC26 was 85% homologous with human ferritin H. Northern blot analysis using the FC26 cDNA insert as a probe confirmed hybridization to an mRNA species of 1 kb, the known size of ferritin H mRNA. In summary, using the technique of differential plaque filter hybridization, we have identified 4 new genes whose mRNA levels are increased by TSH stimulation of thyroid cells. One of these genes is homologous to human ferritin H.     

Human glycophorin A and B are encoded by separate, single copy genes coordinately regulated by a tumor-promoting phorbol ester

Human glycophorin A belongs to a family of structurally related cell surface glycoproteins which are expressed in erythroid cells. We have recently isolated several human glycophorin A-specific cDNA clones and have determined their nucleotide sequence (Siebert, P. D., and Fukuda, M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1665-1669). In this report by using cDNA and exact sequence synthetic oligodeoxyribonucleotides as hybridization probes, we provide evidence that human glycophorin A and B are encoded by separate and distinct genes present as single copies in the human genome. Furthermore, we show that the expression of the glycophorin A and B genes are coordinately regulated by tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate.     

Systematic screening and expression analysis of the head organizer genes in Xenopus embryos.

We describe here a systematic screen of an anterior endomesoderm (AEM) cDNA library to isolate novel genes which are expressed in the head organizer region. After removing clones which hybridized to labeled cDNA probes synthesized with total RNA from a trunk region of tailbud embryos, the 5' ends of 1039 randomly picked cDNA clones were sequenced to make expressed sequence tags (ESTs), which formed 754 tentative unique clusters. Those clusters were compared against public databases and classified according to similarities found to other genes and gene products. Of them, 151 clusters were identified as known Xenopus genes, including eight organizer-specific ones (5.3%). Gene expression pattern screening was performed for 198 unique clones, which were selected because they either have no known function or are predicted to be developmental regulators in other species. The screen revealed nine possible organizer-specific clones (4.5%), four of which appeared to be expressed in the head organizer region. Detailed expression analysis from gastrula to neurula stages showed that these four genes named crescent, P7E4 (homologous to human hypothetical genes), P8F7 (an unclassified gene), and P17F11 (homologous to human and Arabidopsis hypothetical genes) demarcate spatiotemporally distinct subregions of the AEM corresponding to the head organizer region. These results indicate that our screening strategy is effective in isolating novel region-specific genes.     

Characterization of a cluster of human high/ultrahigh sulfur keratin-associated protein genes embedded in the type I keratin gene domain on chromosome 17q12-21

Low stringency screening of a human P1 artificial chromosome library using a human hair keratin-associated protein (hKAP1.1A) gene probe resulted in the isolation of six P1 artificial chromosome clones. End sequencing and EMBO/GenBank(TM) data base analysis showed these clones to be contained in four previously sequenced human bacterial artificial chromosome clones present on chromosome 17q12-21 and arrayed into two large contigs of 290 and 225 kilobase pairs (kb) in size. A fifth, partially sequenced human bacterial artificial chromosome clone data base sequence overlapped and closed both of these contigs. One end of this 600-kb cluster harbored six gene loci for previously described human type I hair keratin genes. The other end of this cluster contained the human type I cytokeratin K20 and K12 gene loci. The center of the cluster, starting 35 kb downstream of the hHa3-I hair keratin gene, contained 37 genes for high/ultrahigh sulfur hair keratin-associated proteins (KAPs), which could be divided into a total of 7 KAP multigene families based on amino acid homology comparisons with previously identified sheep, mouse, and rabbit KAPs. To date, 26 human KAP cDNA clones have been isolated through screening of an arrayed human scalp cDNA library by means of specific 3'-noncoding region polymerase chain reaction probes derived from the identified KAP gene sequences. This screening also yielded four additional cDNA sequences whose genes were not present on this gene cluster but belonged to specific KAP gene families present on this contig. Hair follicle in situ hybridization data for single members of five different KAP multigene families all showed localization of the respective mRNAs to the upper cortex of the hair shaft.     

Cloning and structure of the human interleukin 2 chromosomal gene.

Southern hybridization using 32P-labelled human interleukin 2 (IL2) cDNA probes revealed the existence of a single human IL2 gene. Five clones containing the human IL2 chromosomal gene were isolated from two different human DNA libraries cloned in either lambda Charon 4A or L47 phages. Analysis of the clones showed that they contained different, overlapping portions of human DNA which were derived from the same chromosomal segment. Restriction fragments which hybridized with labelled IL2 cDNA probes were subcloned into plasmid pUR250 and the sequence and organization of the IL2 gene was determined. It contains three introns, 90 bp, +/- 2400 bp and +/- 1900 bp in length, respectively. The organization of the genomic clone resembles that of another lymphokine, interferon-gamma, but no clear homology was found by comparing either the coding sequence or the 5'- and 3'-flanking sequences of the two genes.     

17个新的C2H2型锌指基因片段的分离与克隆

按照Ｃ２Ｈ２型锌指基因保守结构域的ＤＮＡ序列设计一对简并引物,以人基因组ＤＮＡ为模板进行ＰＣＲ同源扩增,将由此获得的锌指基因片段为探针,从人胎肾、骨骼肌、骨骼组织的ｃＤＮＡ分子库中筛选到２２个Ｃ２Ｈ２型锌指蛋白ｃＤＮＡ片段,经国际ＮＣＢＩ数据库查询检索,其中１７个为新的锌指基因片段。对从胎肾ｃＤＮＡ分子库中分离到的Ｋ３－４和Ｋ５－１２克隆进行了表达谱分析,发现Ｋ３－４在肾脏中的表达量明显高于其他几     

Characterization of long cDNA clones from human adult spleen. II. The complete sequences of 81 cDNA clones.

To accumulate information on the coding sequences (CDSs) of unidentified genes, we have conducted a sequencing project of human long cDNA clones. Both the end sequences of approximately 10,000 cDNA clones from two size-fractionated human spleen cDNA libraries (average sizes of 4.5 kb and 5.6 kb) were determined by single-pass sequencing to select cDNAs with unidentified sequences. We herein present the entire sequences of 81 cDNA clones, most of which were selected by two approaches based on their protein-coding potentialities in silico: Fifty-eight cDNA clones were selected as those having protein-coding potentialities at the 5'-end of single-pass sequences by applying the GeneMark analysis; and 20 cDNA clones were selected as those expected to encode proteins larger than 100 amino acid residues by analysis of the human genome sequences flanked by both the end sequences of cDNAs using the GENSCAN gene prediction program. In addition to these newly identified cDNAs, three cDNA clones were isolated by colony hybridization experiments using probes corresponding to known gene sequences since these cDNAs are likely to contain considerable amounts of new information regarding the genes already annotated. The sequence data indicated that the average sizes of the inserts and corresponding CDSs of cDNA clones analyzed here were 5.0 kb and 2.0 kb (670 amino acid residues), respectively. From the results of homology and motif searches against the public databases, functional categories of the 29 predicted gene products could be assigned; 86% of these predicted gene products (25 gene products) were classified into proteins relating to cell signaling/communication, nucleic acid management, and cell structure/motility.     

cDNA sequence, interspecies comparison, and gene mapping analysis of argininosuccinate lyase

A cDNA clone of the argininosuccinate lyase gene (ASL) was isolated from an adult human liver library by probing with synthetic oligonucleotide probes. This clone and a yeast genomic DNA fragment containing the ASL gene were sequenced using the M13-dideoxynucleotide method. Comparison of the yeast and human clones at the nucleotide and putative amino acid sequence levels indicated identities of 50 and 54%, respectively. The most conserved region of the yeast gene was used to detect human clones in the liver cDNA library to test phylogenetic screening capabilities of conserved genes. ASL was mapped to human chromosome 7pter----q22 using human-mouse somatic cell hybrid DNA and further mapped by in situ hybridization to chromosome 7cen----q11.2 on human metaphase chromosomes. The probe also detected a sequence on chromosome 22. Somatic cell hybrid DNA digested with PvuII revealed a mouse polymorphism between Balb/c and C3H mice in the ASL gene.