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PubMed2004

Predicted role for the archease protein family based on structural and sequence analysis of TM1083 and MTH1598, two proteins structurally characterized through structural genomics efforts.

Recently, the structures of two proteins belonging to the archease family, TM1083 from Thermotoga maritima and MTH1598 from Methanobacterium thermoautotrophicum, have been solved independently by two Protein Structure Initiative structural genomics pilot centers using X-ray crystallography and NMR, respectively. The archease protein family is a good example of one of the paradoxes of structural genomics: Approximately one third of protein structures produced by structural genomics centers have no known function and are still annotated as "hypothetical proteins" in the Protein Data Bank. In the case of archeases, despite the existence of two protein structures and abundant sequence information, there is still no function assigned to this protein family. Here, our group predicts, based on structural similarity, sequence conservation, and gene context analyses, that members of this protein family might function as chaperones or modulators of proteins involved in DNA/RNA processing. The conservation of genomic context for this protein family is constant from Archaea and Bacteria to humans, and suggests that unannotated open reading frames contiguous to them could be novel RNA/DNA binding proteins.

Amino Acid Motifs↗
PubMed1994

Characterization of the genomic structure, chromosomal location and promoter of human prostaglandin H synthase-2 gene.

Prostaglandin H synthase (PGHS) is the rate-limiting enzyme in the conversion of arachidonic acid to prostanoids. The human PGHS has two isoforms. PGHS-1 is a house keeping gene whereas PGHS-2 is an inducible gene. We reported here the isolation of the entire PGHS-2 gene and its 5'-flanking region from a human bacteriophage P1 genomic library. The gene containing 10 exons is 7.5 kb in length and located at chromosome 1. The transcriptional start site was mapped at 134 bases upstream from the ATG start codon. Nucleotide sequence of 1.8 kb promoter region contains a TATA box and a number of potential regulatory elements including CRE, NF-kappa B, Sp1 and AP2 sites. Studies of the promoter activity showed that the first 460 nucleotides of 5'-flanking region efficiently drove transcription of the luciferase reporter gene in human umbilical vein endothelial cells upon stimulation with phorbor ester.

Animals↗
PubMed1995

Characterization of the genomic structure and the promoter region of the human intestinal trefoil factor.

Trefoil proteins form a specific group of stable secretory polypeptides. They are expressed in a lot of human cancers and inflammatory processes of the gastrointestinal tract. Recently a new human trefoil protein, ITF/hP1.B was isolated. This gene is expressed mainly in globet cells of intestinum and colon. The genomic structure of the murine and rat homologues genes were described previously and a high nucleotide sequence conservation (78-95%) was found in the coding as well as the regulatory regions. We have isolated the human genomic ITF-gene region from a P1 library and a detailed analysis yielded in a similar gene structure with a three basepair displacement of the second exon intron boundary. The promoter region of the human gene interestingly is markedly different from rat and mouse.

Amino Acid Sequence↗
PubMed1997

Characterization of the genomic structure and promoter of the mouse NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene.

The mouse NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) gene and its 5'-flanking region was cloned from a 129 mouse ES bacteriophage P1 genomic library. The gene contains 7 exons and 6 introns and is 11.3 kb in length. The transcription initiation site was mapped at 35 bases upstream from the ATG start codon. The nucleotide sequence of the 1.6 kb promoter region contains two TATA boxes and a number of potential regulatory elements including Sp1, CRE, GRE, AP1, AP2, NF-IL6 and estrogen receptor binding site. Studies of the promoter's activity showed that the first 400 nucleotides of 5'-flanking region efficiently drove the transcription of the luciferase reporter gene in U936 cells upon stimulation with a phorbol ester.

Animals↗
PubMed1999

Isolation and characterization of a genomic region upstream from the ovine Na+/d-glucose cotransporter (SGLT1) cDNA.

d-Glucose and non-metabolisable analogues of D-glucose regulate the expression of intestinal SGLT1 at both transcriptional and post-transcriptional levels. In order to investigate the molecular mechanisms involved in the transcriptional regulation of the ovine intestinal SGLT1 gene, we have isolated an upstream element of about 1 kb in size. This DNA fragment contains a TATA box motif, 48 bp upstream of the transcriptional start site and includes transcription factor binding sites for HNF-1 and AP-2. We have shown that the ovine SGLT1 promoter fragment can drive the transcription of a reporter gene when transfected into the epithelial cell lines STC-1 and LLC-PK1, which endogenously express SGLT1. Deletion analyses of the promoter indicate that -66/+21 bp proximal sequence directs the highest level of reporter gene activity. There are one and possibly two sites of transcriptional suppression.

5' Untranslated Regions↗
PubMed2000

Genomic organization and characterization of human PEX2 encoding a 35-kDa peroxisomal membrane protein.

Peroxins are proteins involved in peroxisome biogenesis and are encoded by PEX genes. The human PEX2 gene encodes a 35-kDa peroxisomal integral membrane protein which is a member of the zinc finger protein family. Mutations in the PEX2 gene are the primary defect in a subset of patients with Zellweger syndrome and related peroxisome biogenesis disorders. The role of zinc finger proteins in peroxisome assembly and function is poorly understood. Here we report the cloning and characterisation of the human PEX2 structural gene. PEX2 was assigned to human chromosome 8q13-q21 and its murine homologue to mouse chromosome 3. The gene is approximately 17.5 kb in length, and contains four exons. The entire coding sequence is included in one exon, exon 4. The 5'-flanking region has features of housekeeping genes (GC enrichment, two Sp1 sites) and tissue-specific, inducible genes (two CCAAT boxes). In more than 1.5 kb of 5'-flanking sequences we did not identify consensus peroxisomal proliferator responsive elements (PPRE).

Animals↗
PubMed2000

The brain link protein-1 (BRAL1): cDNA cloning, genomic structure, and characterization as a novel link protein expressed in adult brain.

We report here molecular cloning and expression analysis of the gene for a novel human brain link protein-1 (BRAL1) which is predominantly expressed in brain. The predicted open reading frame of human brain link protein-1 encoded a polypeptide of 340 amino acids containing three protein modules, the immunoglobulin-like fold and proteoglycan tandem repeat 1 and 2 domains, with an estimated mass of 38 kDa. The brain link protein-1 mRNA was exclusively present in brain. When analyzed during mouse development, it was detected solely in the adult brain. Concomitant expression pattern of mRNAs for brain link protein-1 and various lectican proteoglycans in brain suggests a possibility that brain link protein-1 functions to stabilize the binding between hyaluronan and brevican. The human BRAL1 gene contained 7 exons and spanned approximately 6 kb. The entire immunoglobulin-like fold was encoded by a single exon and the proteoglycan tandem repeat 1 and 2 domains were encoded by a single and two exons, respectively. The deduced amino acid sequence of human brain link protein-1 exhibited 45% identity with human cartilage link protein-1 (CRTL1), previously reported as link protein to stabilize aggregates of aggrecan and hyaluronan in cartilage. These results suggest that brain link protein-1 may have distinct function from cartilage link protein-1 and play specific roles, especially in the adult brain.

Adult↗
PubMed1996

Cathepsin K: isolation and characterization of the murine cDNA and genomic sequence, the homologue of the human pycnodysostosis gene.

Cathepsin K(EC 3.4.22.38) is a lysosomal cysteine protease that is strongly implicated in bone resorption. The human cathepsin K gene is highly expressed in osteoclasts and gene mutations cause pycnodysostosis, an autosomal recessive skeletal dysplasia. To investigate the evolutionary relatedness of cathepsin K across species, the mouse cathepsin K gene was isolated. A mouse heart cDNA clone, pMCatKl, contained the 3' untranslated region, mature enzyme coding sequence, and most of the propeptide. The remainder of the gene was amplified from mouse melanocyte RNA using 5' rapid amplification of cDNA ends. The gene contained a 990-bp open reading frame, predicting a 329-amino-acid prepropolypeptide. The structure of the protein included a 15-amino-acid presignal, a 99-amino-acid proregion, and a 215-amino-acid mature enzyme. Two potential N-glycosylation sites were identified, one in the proregion and one in the mature enzyme. The 5' untranslated region was 135 bp. The 3' untranslated region was 470 bp including a 9-bp poly(A) tract and contained two polyadenylation signals. The mouse cathepsin K nucleotide and amino acid sequences were highly conserved with the human, rabbit, and chicken homologues across the proregion and mature enzyme. The mouse cathepsin K gene was isolated from an V129 genomic library, and characterization of its genomic structure and intron sizes revealed exons with the initiation ATG in exon 2 and termination TGA in exon 8, a genomic organization that was highly conserved with its human homologue. The availability of the mouse cathepsin K cDNA and genomic sequences will facilitate generation of a mouse model of cathepsin K deficiency by gene targeting.

Amino Acid Sequence↗
PubMed1998

Genomic cloning, molecular characterization, and functional analysis of human CLCA1, the first human member of the family of Ca2+-activated Cl- channel proteins.

We have cloned and molecularly and functionally characterized the first human member of the family of Ca2+-activated Cl- channels, human (h) CLCA1. The 31,902-bp gene is located on chromosome 1p22-31 and is preceded by a canonic promoter region that contains an L1 transposable element. In contrast to all previously known homologs in other species, hCLCA1 is exclusively expressed in intestinal basal crypt epithelia and goblet cells, suggesting that it does not represent the human counterpart of any of them. Expression of the 914-amino-acid hCLCA1 protein in HEK 293 cells yielded a 125-kDa precursor that was processed to yield two cell-surface-associated subunits, a 90-kDa protein and a group of 37- to 41-kDa proteins. Four transmembrane domains were established within the 90-kDa subunit. HEK 293 cells transfected with CLCA1 exhibited an increase in whole-cell Ca2+-sensitive Cl- currents that were outwardly rectified and inhibited by 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid, dithiothreitol, and niflumic acid. Cell-attached patch recordings of transfected cells revealed single channels with a slope conductance of 13.4 pS. These findings suggest that human CLCA1 mediates a Ca2+-activated Cl- conductance in the human intestine and make it an interesting candidate as a modulating factor in the pathogenesis of cystic fibrosis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2001

Molecular characterization of the genome of Maize rayado fino virus, the type member of the genus Marafivirus.

The complete nucleotide sequence of the single-stranded RNA genome of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus, is 6305 nucleotides (nts) in length and contains two putative open reading frames (ORFs). The largest ORF (nt 97-6180) encodes a polyprotein of 224 kDa with sequence similarities at its N-terminus to the replication-associated proteins of other viruses with positive-strand RNA genomes and to the papainlike protease domain found in tymoviruses. The C-terminus of the 224-kDa ORF also encodes the MRFV capsid protein. A smaller, overlapping ORF (nt 302-1561) encodes a putative protein of 43 kDa with unknown function but with limited sequence similarities to putative movement proteins of tymoviruses. The nucleotide sequence and proposed genome expression strategy of MRFV is most closely related to that of oat blue dwarf virus (OBDV). Unlike OBDV, MRFV RNA does not appear to contain a poly(A) tail, and it encodes a putative second overlapping open reading frame.

Amino Acid Sequence↗
PubMed1979

Molecular characterization of the genomes of actinophages SH3, SH10, SH11, and SH12 infecting Streptomyces hygroscopicus.

Some physico-chemical properties of the DNAs released from the actinophages SH3, SH10, SH11, and SH12 are described. The four phage DNAs have a linear double-stranded secondary structure and are unique with respect to their high G.C contents which, from melting studies and buoyant density experiments, were found to be in the range of 68-73 mol-%. The DNA molecular weights were determined by sedimentation velocity experiments and by electron microscopic length measurements, the mean values of the two corresponding data sets being 34.0 x 10(6) (SH3), 26.7 x 10(6) (SH10), 26.1 x 10(6) (SH11), and 28.7 x 10(6) (SH12) with a mean relative error of +/- 5%. From different observations it was concluded that SH10 DNA, and possibly also SH11 and SH12 DNA, have cohesive ends and can undergo intramolecular or intermolecular association to form ring-like monomers or linear and ring-like multimers. Cleavage of the DNAs of SH3, SH10, SH11, and SH12 by EcoRI restriction endonuclease delivered two, one, zero, and two cleavage sites, respectively, and by BamHI restriction endonuclease eight, zero, zero, and zero cleavage sites, respectively.

Bacteriophages↗
PubMed1993

Isolation and characterization of genome-specific DNA sequences in Triticeae species.

Two contrasting genome-specific DNA sequences were isolated from Aegilops speltoides (wild goat grass) and Hordeum chilense (wild barley), each representing more than 1% of the genomes. These repetitive DNA fragments were identified as being genome-specific before cloning by genomic Southern hybridization (using total genomic DNA as a probe), and hence extensive screening of clones was not required. For each fragment, up to six recombinant plasmid clones were screened and about half were genome-specific. Clone pAesKB52 from Ae. speltoides was a 763 bp EcoRI fragment, physically organized in simple tandem repeats and shown to localize to sub-telomeric chromosome regions of species with the Triticeae S-genome by in situ hybridization to chromosomes. The sequence data showed an internal duplication of some 280 bp, which presumably occurred before sequence amplification and dispersion, perhaps by unequal crossing-over or reciprocal translocation. In situ hybridization showed that the sequence distribution varied between closely related (S-genome) species. Clone pHcKB6 was a 339 bp DraI fragment from H. chilense, also tandemly repeated but more variable; loss of the DraI site resulting in a ladder pattern in Southern blots which had little background smear. In situ hybridization showed that the tandem repeats were present as small clusters dispersed along all chromosome arms except at a few discrete regions including the centromeres and telomeres. The clone hybridized essentially specifically to the H-genome of H. chilense and hence was able to identify the origin of chromosomes in a H. chilense x Secale africanum hybrid by in situ hybridization.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMedDate not supplied

The presence of a nucleomorph hsp70 gene is a common feature of Cryptophyta and Chlorarachniophyta.

Cryptomonad algae and Chlorarachniophyta are evolutionary chimaeras derived from the engulfment of an eukaryotic phototrophic endosymbiont by a eukaryotic host cell. Although much reduced, the endosymbiont's eukaryotic plasmatic compartment still contains a nucleus, the so-called nucleomorph. These nucleomorphs carry the smallest known eukaryotic genomes. We have characterized the genomes of several cryptomonads and a Chlorarachnion species by means of PFGE (pulsed-field gel electrophoresis). Hybridization studies with small subunit rDNA were used to identify the nucleomorph chromosomes. We also performed hybridization experiments with an hsp70 probe to estimate the distribution of this gene among the different algal species. The evolutionary, genetical, and physiological implications of our studies are discussed. A model on the possible function of the nucleomorph hsp70 gene products is presented.

Blotting, Southern↗
PubMed1985

Physical characterization of the genome of the Myxococcus xanthus bacteriophage MX-8.

We have constructed a restriction map for the genome of bacteriophage MX-8 from Myxococcus xanthus using the enzymes PvuII, MboI, and EcoRI. The phage genome size, as determined by restriction analysis, is 51.7 +/- 0.6 Kb. Double digestions, redigestions of isolated fragments, and crossed-contact hybridization of partial digestion products show that the restriction map is circular. Restriction analysis and Southern hybridization show that the phage DNA molecules are packaged sequentially from a concatemer starting from a specific site which we have mapped. The DNA molecules have an average terminal redundancy of approximately 8% and are circularly permuted over at least 40% of the genome.

Bacteriophages↗
PubMed1988

Characterization of the genome of a vaccine strain of canine adenovirus type 1.

Restriction endonuclease cleavage maps have been constructed for the genome of a canine adenovirus type 1 (CAV-1) vaccine strain (CLL; Connaught Laboratories, Ltd., Willowdale, Ontario). Restriction enzyme analyses were also carried out on CAV-1 (CLL) genomes isolated from viral stocks over 8 serial passages in a dog kidney cell line (DK 6722). The right hand 20% of the genome became more heterogeneous in size with increasing passage in DK 6722 cells due to deletions up to 3-4 kb, whereas the left terminal region was stable throughout these passages. A comparative study of CAV-1(CLL) and a virulent strain of CAV-1, Glaxo, revealed that the genome of CAV-1(CLL) was the shorter, by about 480 bp, within the region covering 0.83-0.91 map units. By virtue of its location within the genome and its dispensable nature for viral growth, this region would appear to encompass a genetic sequence corresponding to the E3 region of human adenoviruses. In terms of viral attenuation, the possible importance of the observed differences between CAV-1(CLL) and CAV-1(Glaxo) is discussed.

Adenoviruses, Canine↗
PubMed2003

Characterization of soybean genomic features by analysis of its expressed sequence tags.

We analyzed 314,254 soybean expressed sequence tags (ESTs), including 29,540 from our laboratory and 284,714 from GenBank. These ESTs were assembled into 56,147 unigenes. About 76.92% of the unigenes were homologous to genes from Arabidopsis thaliana ( Arabidopsis). The putative products of these unigenes were annotated according to their homology with the categorized proteins of Arabidopsis. Genes corresponding to cell growth and/or maintenance, enzymes and cell communication belonged to the slow-evolving class, whereas genes related to transcription regulation, cell, binding and death appeared to be fast-evolving. Soybean unigenes with no match to genes within the Arabidopsis genome were identified as soybean-specific genes. These genes were mainly involved in nodule development and the synthesis of seed storage proteins. In addition, we also identified 61 genes regulated by salicylic acid, 1,322 transcription factor genes and 326 disease resistance-like genes from soybean unigenes. SSR analysis showed that the soybean genome was more complex than the Arabidopsis and the Medicago truncatula genomes. GC content in soybean unigene sequences is similar to that in Arabidopsis and M. truncatula. Furthermore, the combined analysis of the EST database and the BAC-contig sequences revealed that the total gene number in the soybean genome is about 63,501.

Chromosomes, Artificial, Bacterial↗