Sequence of human villin: a large duplicated domain homologous with other actin-severing proteins and a unique small carboxy-terminal domain related to villin specificity.

Villin is a calcium-regulated actin-binding protein that caps, severs, and bundles actin filaments in vitro. This 92,500-D protein is a major constituent of the actin bundles within the microvilli of the brush border surface of intestinal and kidney proximal tubule cells. Villin is a very early marker of cells involved in absorption and its expression is highly increased during intestinal cell differentiation. The amino acid sequence deduced from the cDNA sequence revealed that human villin is composed of three domains. The first two domains appear as the result of a duplication: their structural organization is similar. We can then define a basic unit in which a slightly hydrophilic motif is followed by three hydrophobic motifs, similar between themselves and regularly spaced. The duplicated domain is highly homologous to three other actin-severing proteins and this basic structure represents the whole molecule in severin and fragmin, while two basic units compose gelsolin. The third domain which is carboxy terminal is villin specific: it is unique among actin modulating proteins so far known. It could account for its actin-binding properties (dual regulation by calcium of severing and bundling activities). We propose that it may also be related to the subcellular localization of villin in different epithelial cell types.

ICBP90, a novel human CCAAT binding protein, involved in the regulation of topoisomerase IIalpha expression.

The one-hybrid system with an inverted CCAAT box as the DNA target sequence was used to identify proteins acting on key DNA sequences of the promoter of the topoisomerase IIalpha gene. Screening of cDNA libraries from the leukemia Jurkat cell line and from the adult human thymus resulted in the isolation of a novel protein of 793 amino acids (89,758 Da). This protein has in vitro CCAAT binding properties and has been called ICBP90. Adult thymus, fetal thymus, fetal liver, and bone marrow, known as active tissues in terms of cell proliferation, are the tissues richest in ICBP90 mRNA. In contrast, highly differentiated tissues and cells such as the central nervous system and peripheral leukocytes are free of ICBP90 mRNA. Western blotting experiments showed a simultaneous expression of topoisomerase IIalpha and ICBP90 in proliferating human lung fibroblasts. Simultaneous expression of both proteins has also been observed in HeLa cells, but in both proliferating and confluent cells. Overexpression of ICBP90 in COS-1-transfected cells induced an enhanced expression of endogenous topoisomerase IIalpha. Immunohistochemistry experiments showed that topoisomerase IIalpha and ICBP90 were coexpressed in proliferating areas of paraffin-embedded human appendix tissues and in high-grade breast carcinoma tissues. We have identified ICBP90, which is a novel CCAAT binding protein, and our results suggest that it may be involved in topoisomerase IIalpha expression. ICBP90 may also be useful as a new proliferation marker for cancer tissues.

Characterization of the biotin biosynthesis pathway in Saccharomyces cerevisiae and evidence for a cluster containing BIO5, a novel gene involved in vitamer uptake.

An engineered mutant of Saccharomyces cerevisiae affected in biotin biosynthesis has been isolated. This mutant allowed the characterization of a bio cluster (BIO3-4-5). We demonstrate that BIO3 (YNR058w) and BIO4 (YNR057c) encode, respectively, a 7, 8-diaminopelargonic acid aminotransferase and a dethiobiotin synthase, involved in the biotin biosynthesis pathway. A novel gene, BIO5 (YNR056c), is present immediately downstream from BIO4. This gene encodes Bio5p, a protein with 11 putative transmembrane regions. Uptake experiments performed with labeled 7-keto 8-aminopelargonic acid indicate that Bio5p is responsible for transport into the cell of 7-keto 8-aminopelargonic acid.

Chicken poly(ADP-ribose) synthetase: complete deduced amino acid sequence and comparison with mammalian enzyme sequences.

The complete nucleotide (nt) sequence of the cDNA encoding the chicken poly(ADP-ribose) synthetase has been determined. Positive clones overlapping the 5' region or the 3' region of the cDNA have been isolated from a lambda gt 10 hen oviduct cDNA library using two human cDNA probes. The missing middle portion has been obtained by the polymerase chain reaction procedure. A single 3033-nt open reading frame from start codon to stop codon encodes a sequence of 1011 amino acid residues. The alignment of this sequence with those from human and mouse reveals overall identities of 79% and 77%, respectively. However, an identity of about 82% is obtained in the DNA-binding domain within the two zinc fingers, and an even higher similarity (85-87%) is observed in the NAD-binding domain. The isolated clones consistently hybridize on chicken Northern blots to an mRNA species of about 4 kb, whereas they do not cross-hybridize with RNA blots of Drosophila melanogaster. Thus, it appears that, even if the functional properties of the enzyme are maintained, the cDNA identity will be much decreased in nonvertebrate organisms.

Complete structure of the human transferrin gene. Comparison with analogous chicken gene and human pseudogene.

The complete structure of the human transferrin gene is presented. This gene has a total size of about 33.5 kb and is organized in 17 exons separated by 16 introns. The chicken ovotransferrin gene has a size of 10.5 kb and is also organized in 17 exons and 16 introns. The analysis of the structure of the two genes confirm, at the gene level, that transferrins originated by a gene duplication phenomenon. Finally, the existence of a new member of the transferrin family, a human transferrin non-processed pseudogene is demonstrated.

Isolation and characterization of an expressed hypervariable gene coding for a breast-cancer-associated antigen.

A human gene and cDNA coding for a breast-cancer-associated antigen (H23Ag) were isolated and characterized. The gene contains two exons and one intron. Part of the second exon is a tandem repeat array (TRA) consisting of multiple 60-bp G + C-rich units. We report here the characterization of unique sequences that are found in the H23Ag gene and cDNA, in addition to the 60-bp repeats. Analysis of the cDNA sequences revealed a putative ATG start codon preceded by two overlapping initiation consensus sequences (CCACC). The open reading frame determines an amino acid (aa) sequence consisting of three regions. The first region contains an initiating methionine and a highly hydrophobic putative signal peptide. This is followed by a variable number of highly conserved 20-aa repeat units (TRA). The last region, C-terminal to TRA, contains four potential N-linked glycosylation sites. The genomic nucleotide sequences demonstrate a putative promoter region that includes a 'TATA' box. A putative estrogen regulatory element is located 5' to the promoter region. The characterization of the gene and cDNA coding for the H23Ag presented here, may help to elucidate its possible function in human breast cancer.

Routine detection of Salmonella species in water: comparative evaluation of the ISO and PROBELIA polymerase chain reaction methods.

Water is one of the main transmission routes for Salmonella spp., the causative agents of salmonellosis in humans and animals. This worldwide sanitary problem requires rapid and accurate analyses to be realized so that advisories for exposed people are timely and reliable. Because the traditional method, the ISO 6340 method, does not meet the criterion of rapidity, there was a need for a quicker method. To fulfill this need, a comparative evaluation between the ISO method and one based on polymerase chain reaction (PCR), the PROBELIA, was performed. Waters from different origins were tested either directly or after artificial contamination with selected Salmonella strains isolated from the environment. The results clearly demonstrate that the PCR-based method can advantageously replace the ISO 6340 method. It is quicker, less labor intensive, reproducible, and provides results that match perfectly to those obtained by the ISO method. Furthermore, the PROBELIA method meets the requirements for an efficient sanitary survey: high numbers of samples processed at the same time, reduced cost, and results within 2 days.

The complete nucleotide sequence of the chicken ovotransferrin mRNA.

The nucleotide sequence of an almost double-stranded cDNA copy [Cochet, M., Perrin, F., Gannon, F., Krust, A., Chambon, P., McKnight, G. S., Lee, D. C., Mayo, K. E., and Palmiter, R. D. (1979) Nucleic Acids Res. 6, 2435-2452] of chicken ovotransferrin (conalbumin) mRNA has been determined. Taking into account the previously reported 5'-end sequence [Cochet, M., Gannon, F., Hen, R., Maroteaux, L., Perrin, F., and Chambon, P. (1979) Nature (Lond.) 282, 567-574] we present the complete nucleotide sequence of the ovotransferrin mRNA from which the amino acid sequence of the protein is inferred. A computer and statistical analysis of the nucleotide sequence reveals a pattern of internal homology which confirms that the present-day chicken ovotransferrin gene (and by extrapolation the transferrin genes of other species) has evolved by duplication and gives some support to the quadruplication hypothesis of transferrin evolution.

The 5' flanking region of the human pS2 gene mediates its transcriptional activation by estrogen in MCF-7 cells.

The pS2 gene is transcriptionally induced by oestrogen in the human breast cancer cell line MCF-7. We demonstrate here that the 5' flanking sequences (-3000 to +10 bp) of the pS2 gene possess the properties of an oestrogen-inducible promoter. Interestingly, this oestrogen induction could not be demonstrated in transient transfection assays in MCF-7 cells, but only in stably transformed MCF-7 cells, which suggests that some factors responsible for oestrogen induction may be present in limiting amounts in these cells and absent in HeLa cells.

Characterization of a gene encoding a 115 K super T antigen expressed by a SV40-transformed rat cell line.

It has been reported that SV40-transformed V 11 F 1 clone 1 subclone 7 rat cells (subclone 7) produce a super T antigen of 115,000 M. This super T antigen is entirely SV40 coded and is synthesized by translation of an elongated form of SV40 early mRNA (May, E., Kress, M. Daya-Grosjean, L., Monier, R. and May, P. (1981) J. Virol., 37, 24-35). The results reported here show that there is only one independent insertion of viral DNA in the cellular genome of subclone 7 cells. When DNA from subclone 7 cells was cleaved with Bam HI endonuclease two distinct SV40 sequence containing fragments were generated with sizes of 5 Kb and 10 Kb, respectively. Two recombinant cosmids were constructed by insertion of the 5 Kb and 10 Kb fragments, respectively, into cosmid pHC 79. Using restriction map analysis and nucleotide sequencing, we showed that the 5 Kb fragment actually contained the complete sequence of a gene encoding super T antigen. As compared to the normal SV40 early gene, the sequence of super T gene showed the following rearrangements: (i) The segment between nucleotides 4116 - 3544 was duplicated in a direct order and (ii) these two copies of 573 nucleotide sequence were separated by a 93 nucleotide tract which was a nearly perfect inverted repeat of the segment located between nucleotides 4868 and 4776 (nucleotide numbering used here = Weissmann number +17).

Cloning of Schizosaccharomyces pombe bio2 by heterologous complementation of a Saccharomyces cerevisiae mutant.

A Saccharomyces cerevisiae mutant affected in the last step of the biotin biosynthesis pathway was isolated by using a transposon mutagenesis method. The gene BIO2, encoding a biotin synthase, is shown to be interrupted in this mutant. Heterologous complementation experiment allowed the cloning and the characterization of a novel bio gene: bio2, encoding biotin synthase from Schizosaccharomyces pombe.

A detailed comparison of the 5'-end of the ovalbumin gene cloned from chicken oviduct and erythrocyte DNA.

We have examined homologous fragments of DNA cloned from two different tissues for changes in the dNA sequence which might be related to tissue specific gene expression. The 5' end of the chicken ovalbumin gene was cloned from oviduct or erythrocyte DNA DNA using cosmids as vectors. We have compared the two clones obtained by restriction enzyme digestions, analysis of heteroduplexes by electron microscopy or S1 nuclease digestion and by DNA sequencing. Our results show that whereas no alteration occured in the region of the gene assumed to be of importance for the control of transcription, a 4 nucleotide deletion/insertion was detected in the first intron of the ovalbumin gene.

Activation of pS2 gene transcription is a primary response to estrogen in the human breast cancer cell line MCF-7.

We have shown previously that an increase in the level of accumulated pS2 mRNA is first detectable in MCF-7 cells after 3 hr of estradiol treatment. Using in vitro nuclear run-on transcription with nuclei prepared from MCF-7 cells grown in the presence of estradiol or in estradiol-stripped medium, we demonstrate here that expression of the pS2 gene is controlled by estrogen at the transcriptional level. Induction of transcription is a very early event that is already apparent within 15 min after addition of estradiol to the culture medium. In addition, pretreatment of the cells with the protein synthesis inhibitor cycloheximide does not prevent induction of pS2 gene transcription, indicating that it corresponds to a primary effect of estrogen. The pS2 gene in MCF-7 cells represents a unique example of a human gene whose transcription is directly controlled by estrogen.

In situ mapping of the chicken progesterone receptor gene and the ovalbumin gene.

The chicken progesterone receptor (cPR) gene and the ovalbumin (OA) gene, a target of cPR regulation, have been mapped via fluorescent in situ hybridization to the two largest chromosomes of the chicken karyotype. cPR is subtelomeric on the long arm of chromosome 1 and OA is on the long arm of chromosome 2, close to the centromere. A 35-kb cosmid probe for the cPR gene and two genomic fragments of 9.2 and 15 kb for the OA gene were biotin-labeled for nonradioactive localization of the two chicken loci.

Isolation of high-molecular-weight DNA from eukaryotic cells by formamide treatment and dialysis.

A new method for the isolation of high-molecular-weight DNA from eukaryotic cells is presented. It is based on formamide treatment and extensive dialysis of cellular proteinase K digests. This procedure consists mainly of a series of incubations allowing simultaneous preparation of several DNAs which can then be restricted, ligated, hybridized, and cloned.

Microchromosomal assignment of the chicken ovotransferrin and adenylate kinase genes.

Ovotransferrin, an egg-white protein implicated in the transfer of trace elements from the hen oviduct to the developing avian embryo, and cytosolic adenylate kinase, an essential enzyme involved in the interconversion of adenine nucleotides in energetically active tissues, have been mapped to two separate chicken microchromosomes by fluorescent in situ hybridization. Considering present and previous data, the possibility of loss of intron material resulting in the compactation of genes in chicken microchromosomes is briefly discussed.

A DNA recombinant database management system.

A set of computer programs is described which constitutes a clone database management system. Maintenance of the database and the stocks of material is designed to be under the control of one person or group of people, who may insert, delete or modify data entries, and who may interrogate the database as to which stocks are in need of checking. The system is organised in such a way that information is freely and speedily available to all users. Database entries may be accessed by name or key word.

Sequence of the pS2 mRNA induced by estrogen in the human breast cancer cell line MCF-7.

We present the complete sequence of an mRNA which is induced by estrogen in the human breast cancer cell line MCF-7 [pS2 mRNA, Masiakowski et al., Nucleic Acids Res. 10, 7895-7903 (1982)]. Primer extension and cloning of double-stranded cDNA (ds-cDNA) into a vector designed to make full-length cDNA were used to determine the sequence of the fifteen 5'-terminal nucleotides which were not present in the original pS2 ds-cDNA clone. The mRNA sequence has a major open reading frame encoding 84 amino-acids, flanked by a 40 nucleotide 5'-untranslated region and a 198 nucleotide 3'-untranslated region preceding the polyA tail. The 3'-untranslated region contains a polyadenylation signal, AUUAAA, 14 nucleotides upstream from the polyA tail. The derived protein sequence contains a putative signal peptide region suggesting that the protein may be secreted. The nucleotide and derived amino-acid sequences were compared to previously determined sequences, particularly to those of hormone-regulated proteins and growth factors, and no obvious similarities were observed.

Crystallization of the 43 kDa ATPase domain of Thermus thermophilus gyrase B in complex with novobiocin.

The 43 kDa ATPase domain of Thermus thermophilus gyrase B was overproduced in Escherichia coli and a three-step purification protocol yielded large quantities of highly purified enzyme which remained stable for weeks. Crystals of the 43 kDa domain in complex with novobiocin, one of the most potent inhibitors of bacterial topoisomerases, were obtained. Crystals obtained in the presence of PEG 8000 do not diffract, but a different crystal form was obtained using sodium formate as a precipitating agent. The plate-shaped crystals, which were less than 10 microm in thickness, could be cryocooled directly from the mother liquor and a full diffraction data set was collected to 2.3 A allowing the determination of the first structure of a gyrase B 43K domain in complex with a coumarin.