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At least 829 records · Page 46Linked to original sources
PubMed1997

Characterization and regulation of the genes encoding ribosomal proteins L39 and S7 of the human pathogen Candida albicans.

Genes encoding the Candida albicans ribosomal proteins L39 and S7 (RPL39, RPS7) were isolated and sequenced. From RPL39 cDNA a single intron interrupting the fifth codon in the genomic sequence could be deduced. Two homologous RPL39 genes in Saccharomyces cerevisiae contain a single intron in a conserved position. In contrast, C. albicans RPS7 was found to lack an intron, while both S. cerevisiae homologs are interrupted by single introns. The deduced L39 and S7 proteins contained 67% and 83% identical residues compared to the S. cerevisiae homologs. During hyphal induction the RPL39, RPS7 and RPL29 transcript levels increased three- to six-fold relative to ribosomal RNA, while ACT1 and RPS33 control transcripts were not regulated extensively. As suggested by unaltered transcript stabilities during hyphal induction, this regulation occurs on the transcriptional level; a conserved 18 bp palindromic sequence (5'-TTAGGGCTATAGCCCTAA-3'), which is present in the promoter regions of the RPL39 and RPS7 genes, may be involved in regulation.

Amino Acid Sequence↗
PubMed1998

Identification of a putative histidine kinase two-component phosphorelay gene (CaHK1) in Candida albicans.

We have cloned and analysed the sequence of a putative histidine kinase, two-component gene (CaHK1) from Candida albicans. This gene encodes a 2471 amino acid protein (Cahk1p) with an estimated molecular mass of 281.8 kDa. A homology search of Cahk1p with other proteins in the databases showed that Cahk1p exhibits the greatest homology at its C-terminus with both the sensor and regulator components of prokaryotic and eukaryotic two-component histidine kinases. A further analysis of this homology showed that the Cahk1p possessed both sensor and regulator domains in the same polypeptide. Also, Cahk1p is likely to be a soluble protein. The sensor kinase domain of Cahk1p contains conserved motifs that are characteristic of all histidine kinase proteins, including the putative histidine which is believed to be autophosphorylated during activation, ATP binding motifs and others (F- and N-motifs), with unknown function. The Cahk1p regulator domain also contains conserved aspartate and lysine residues and the putative aspartate, which is secondarily phosphorylated by the autophosphorylated histidine. Finally, according to the codon usage frequency of the CaHK1 gene in comparison with other genes from C. albicans, there would appear to be a low level of expression of the gene.

Amino Acid Sequence↗
PubMed1999

unc-45 gene of Caenorhabditis elegans encodes a muscle-specific tetratricopeptide repeat-containing protein.

The unc-45 gene of the nematode, Caenorhabditis elegans, is essential for muscle organization and embryonic development. Genetic evidence suggests the unc-45 gene product controls muscle thick filament assembly. We report here on the determination of the gene's chromosomal location and the isolation and sequencing of its cDNA. The amino terminus of the predicted unc-45 protein contains three tandem repeats that belong in the tetratricopeptide repeat family. Tetratricopeptide motifs have been shown to be involved in protein interactions, and some of the closest homologues have chaperone-like activity. The carboxy terminus of the protein has homology with the related fungal proteins, CRO1 and She4p, which have been postulated to play a role in assembly of or interactions with a cytoplasmic myosin. We have also determined the sequence of the homologous gene from C. briggsae, which demonstrates a high level of conservation. We show that the unc-45 gene promoter can drive reporter gene expression, which is limited to muscle tissues (pharyngeal, body wall, vulval, and anal muscles), consistent with a role for the unc-45 gene in muscle development or function.

Amino Acid Sequence↗
PubMed1996

Cloning, characterization, and expression of the transforming growth factor-beta type I receptor promoter in fetal rat bone cells.

Transforming growth factor (TGF-beta) binds several discrete membrane proteins. Of these, a type 1 receptor appears indispensable for signal transduction. Previous examination of TGF-beta receptor expression has been limited to changes in cell surface protein, and more recently, mRNA abundance. In order to learn more about TGF-beta function and receptor expression during osteogenesis, we have now cloned a 4 kilobase (kb) DNA fragment 5' proximal to the coding region of the rat TGF-beta type I receptor gene. Sequence analysis revealed multiple elements compatible with transcription initiation, including a properly positioned and oriented CCAAT box, six Sp1 binding sites (three defining GC boxes), and two strong AP2 binding sites within a 0.7 kb span directly upstream of the coding region. The 3' terminal 0.3 kb span comprises a GC-enriched (77%) so-called CpG island that, like other similarly organized promoters, lacks a TATA box. Primer extension and RNase protection studies with cRNAs from this area show multiple initiation sites within 220 bp 5' proximal to the initial methionine codon. Transient transfections using nested, deleted, and inverted promoter sequences demonstrated maximal reporter expression by a 1 kb fragment encompassing all of these elements. Truncation of the 1 kb fragment from the 5' and 3' ends indicated the need for several elements for peak promoter activity. These results, and transfections in fetal rat bone and dermal cells, suggest that this promoter contains elements that specify basal and conditional expression of the TGF-beta type I receptor in bone.

Amino Acid Sequence↗
PubMed1999

Comparison of three methods for single nucleotide polymorphism typing for DNA bank studies: sequence-specific oligonucleotide probe hybridisation, TaqMan liquid phase hybridisation, and microplate array diagonal gel electrophoresis (MADGE).

In the near future the number of SNPs identified and mapped will increase and the need for high throughput SNP typing will be paramount for comprehensive examination by association of the role of genomic regions in disease traits. A range of higher throughput methods for typing SNPs is now in routine use in many laboratories worldwide. In this report, we analyse the relative advantages and disadvantages of three such methods, TaqMan, PCR-SSOP, and ARMS-MADGE, currently in use in our laboratories. Throughputs achievable are similar, but there are major differences in cost and time for set-up, equipment, consumables, and staff time, which may determine the choice for individual laboratories.

Costs and Cost Analysis↗
PubMed1997

Genomic organization of the mouse fertilin beta gene that encodes an ADAM family protein active in sperm-egg fusion.

The fertilin alpha and beta genes (Ftna and Ftnb, respectively) encode a sperm surface heterodimer that functions in sperm-egg fusion. They are the first identified members of a large gene family coding for multidomain membrane proteins (ADAMs) that include A Disintegrin And Metalloprotease domain. In this study, we report the isolation and structural organization of the mouse fertilin beta gene. The gene is present as a single copy and covers a region of approximately 55 kilobases in the genome. The fertilin beta gene is composed of at least 20 exons interrupted by 19 introns. The sizes of the exons are relatively small and vary from 56 to 193 bases; the sizes of introns vary from 350 bases to 9.4 kilobases. The exon-intron boundaries conform to the GT/AG rule with one exception: GC replaces GT at the 5' splice site in intron 13. Comparison of genomic organization between mouse fertilin beta and the previously sequenced ADAM family gene, human MDC [Katagiri et al. (1995): Cytogenet Cell Genet 68:39-44] showed 12 conserved exon-intron boundaries. In addition, we analyzed the fertilin alpha gene, demonstrating that more than one gene is present in the mouse genome.

ADAM Proteins↗
PubMed1998

Genetic origin of IgG antibodies cloned by phage display and anti-idiotypic panning from three patients with autoimmune thrombocytopenia.

The beneficial use of intravenous immunoglobulins (IVIG) in certain groups of patients with autoimmune thrombocytopenic purpura (AITP) has been proven. AITP is a severe disease in children with a still unknown etiology. It is not clear how IVIG functions in this and other autoimmune diseases. To analyze and compare patient-derived monoclonal IgG antibodies that are bound by IVIG in an anti-idiotypic manner, the combinatorial antibody phage display system was applied. From three different patients with AITP, a large number of clones specifically reacting with IVIG molecules were enriched. The heavy and light chain variable regions were sequenced and compared with each other and with databases. Many variable regions showed extensive replacement mutations within the complementarity-determining regions, while two were identical to germ-line genes. Our data show that the most frequently used germ-line gene loci of these IVIG binders are identical to those observed for many other autoantibodies. This implicates a specific interaction of IVIG particularly with autoantibodies and B cell receptors derived from germ-line genes that are often used for the generation of autoantibodies.

Adolescent↗
PubMed2001

The Cancer Genome Anatomy Project: new resources for reading the molecular signatures of cancer.

The Cancer Genome Anatomy Project (CGAP) has built informational, technological, and physical resources to interface genomics with basic and clinical cancer research. The CGAP web site (http://cgap.nci.nih.gov) provides informatics tools for in silico analysis of the CGAP datasets as well as information for accessing each of the CGAP resources. Published in 2001 by John Wiley & Sons, Ltd.

Animals↗
PubMed2000

Differential expression and subcellular localization of murine BRCA1 and BRCA1-delta 11 isoforms in murine and human cell lines.

BRCA1 mutations are involved in breast and ovarian cancer predisposition in humans. The biological functions of the murine BRCA1 gene have been extensively studied but little is known about murine BRCA1 proteins. To better characterize these proteins, we have cloned the full-length murine BRCA1 cDNA and a splice variant deleted of exon 11, BRCA1-delta 11, by RT-PCR method. Three polyclonal antibodies raised against various parts of murine BRCA1 were used in our study: D16, M20 and 5MO, which were generated in our laboratory. This allowed us to analyze the expression and subcellular localization of both isoforms in murine and human cell lines by immunoblotting, immunoprecipitation, cell fractionation and immunofluorescence. Endogenous BRCA1 was detected in murine cell lines but not splice variant BRCA1-delta 11, whereas both ectopically expressed murine isoforms were detected in transfected human Bosc 23 cells. Subcellular fractionation and immunofluorescence results showed that the BRCA1 protein was mainly located in the nucleus, whereas BRCA1-delta 11 was preferentially cytoplasmic. The conservation of exon 11 splicing and the differential subcellular localization of BRCA1 and BRCA1-delta 11 in human and mouse suggest that these proteins could play distinct roles and that they could differentially act in the pathological mechanisms leading to the development of breast and ovarian cancer. The characterization of the murine BRCA1 proteins and antibodies will be useful to further study BRCA1 functions in murine models.

3T3 Cells↗
PubMed2002

A human TAPBP (TAPASIN)-related gene, TAPBP-R.

TAPASIN, a V-C1 (variable-constant) immunoglobulin superfamily (IgSF) molecule that links MHC class I molecules to the transporter associated with antigen processing (TAP) in the endoplasmic reticulum (ER) is encoded by the TAPBP gene, located near to the MHC at 6p21.3. A related gene was identified at chromosome position 12p13.3 between the CD27 and VAMP1 genes near a group of MHC-paralogous loci. The gene, which we have called TAPBP-R (R for related), also encodes a member of the IgSF, TAPASIN-R. Its putative product contains similar structural motifs to TAPASIN, with some marked differences, especially in the V domain, transmembrane and cytoplasmic regions. By using the mouse ortholog to screen tissue, we revealed that the TAPBP-R gene was broadly expressed. Sub-cellular localization showed that the bulk of TAPASIN-R is located within the ER but biotinylation experiments were consistent with some expression at thecell surface. TAPASIN-R lacks an obvious ER retention signal. The function of TAPASIN-R will be of interest in regards to the evolution of the immune system as well as antigen processing.

Amino Acid Motifs↗
PubMed2002

The M603 idiotype is lost in the response to phosphocholine in terminal deoxynucleotidyl transferase-deficient mice.

The majority of anti-phosphocholine (PC) antibodies induced by the PC epitope in Proteus morganii (PM) express the M603 idiotype (id), which is characterized by an invariant Asp to Asn substitution at the V(H):D(H) junction. To elucidate the molecular basis by which M603-like B cells acquire the mutations resulting in this invariant substitution, we analyzed the immune response to PC-PM in terminal deoxynucleotidyl transferase (TdT) gene knockout (KO) mice. In the absence of TdT, T15-id antibodies comprised 80-100% of the primary response to PC-PM. Less than 10% of the response in wild-type mice is T15-id(+). In TdT KO mice, the secondary response to PC-KLH was higher than in wild-type mice and was dominated by the germ-line T15-id. About 10% of this response, in both TdT KO and wild-type mice, comprised M167-id(+) antibodies. Additionally, none of the functionally rearranged V1/DFL16.1/J(H)1 cDNA isolated from PC-PM-immunized TdT KO mice showed the Asp/Asn substitution characteristic of PC-binding, PC-PM-induced M603-like antibodies. These data indicate that production of M603-id antibody is TdT dependent, while generation of M167-id antibody is TdT independent, and that in the absence of competition from M603-like B cells, T15-id B cells can respond to PC-PM.

Amino Acid Substitution↗
PubMed2007

Droplets as microreactors for high-throughput biology.

Inspired by the principles of biological evolution, biologists--and others--have in recent decades harnessed the power of "natural" selection to sift through huge libraries of genes and find those with desirable properties. At the same time, the demand for high-throughput biochemical and genetic assays and screens has driven the development of increasingly miniaturised assay systems. An exciting synergy is now emerging between these two fields, whereby the tools of ultrahigh-throughput screening promise to open up new directions in molecular engineering.

Bacteria↗
PubMed2005

Dynamic Id2 expression in the medial and lateral domains of avian dermamyotome.

Id2 cDNA was isolated from a subtractive screen of stage-12 quail caudal somites. In situ hybridisation analysis identified the previously un-described expression of Id2 mRNA in distinct medial and lateral domains of the somitic dermamyotome in both quail and chick embryos. Id2 expression in somites was highly dynamic being first initiated in the lateral domain of the dermamyotome of stage-8-10 embryos, followed by expression in a separate medial domain. Id2 mRNA during subsequent embryonic development could be detected in both medial and lateral domains in the anterior to mid regions while the posterior, recently segmented somites, showed expression only in the lateral domain, which was eventually down regulated in the anterior-most somites. Tissue manipulation studies revealed that Id2 expression in somites required positive signalling from not only axial structures and lateral plate mesoderm but also surface ectoderm. In addition, Id2 expression was also observed in anterior and posterior domains of developing avian limb buds and interdigital tissue.

Animals↗
PubMed1990

Characterization and expression of two sea urchin homeobox gene sequences.

We describe two homeobox sequences, TgHbox5 and TgHbox6, isolated from the Hawaiian sea urchin Tripneustes gratilla using a Drosophila Sex combs reduced probe. Sequence analysis shows that the encoded TgHbox5 homeodomain shares only 30-52% amino acid identity with homeodomains encoded by previously characterized genes, establishing that it is a divergent homeobox that is not in any known class of homeoboxes. TgHbox5 is expressed in the embryo as two major developmentally regulated transcripts. one at 5.0 kilobase (kb) appearing by blastula stage and the other at 2.7 kb appearing at pluteus stage. Multiple transcripts from TgHbox5 are present at a much lower level in adult tissues and are predominantly expressed in small and large intestines. The TgHbox6 homeobox is an Antenna-pedia-class homeobox, which appears not to be expressed during embryogenesis but produces abundant 3.6 and 3.2 kb transcripts in the six adult tissues examined.

Amino Acid Sequence↗
PubMed1995

Immunoglobulin heavy chain germ-line JH-C mu transcription in human precursor B lymphocytes initiates in a unique region upstream of DQ52.

From human precursor B cells which had both immunoglobulin (Ig) heavy (H) chain loci in germ-line configuration, various IgH chain germ-line transcripts were isolated and sequenced. These transcripts were shown to contain sequences derived from the JH region, the IgH chain enhancer element or the Ig switch region. A number of isolated cDNA clones contained sequences at their 5' end that were derived from a single exon located just upstream of DQ52, designated the mu o' element. Sequence analysis of a 920-bp genomic DNA segment, containing the mu o' exon and its 5' flanking region, revealed the presence of various conserved motifs for DNA-binding proteins, such as E2A, Ets, NF-kappa B and AP-2, which have previously been found in the IgH and L chain enhancers. We propose that the activity of the mu o' element, resulting in germ-line transcription of the DQ52-JH gene segment, is required to generate full accessibility for the V(D)J recombinase.

B-Lymphocytes↗
PubMed1995

Evolution of specific antigen recognition: size reduction and restricted length distribution of the CDRH3 regions in the rainbow trout.

The immunoglobulin heavy chain repertoire in fish was investigated by cloning a total of 88 rearranged VDJ junctions from the head kidney B cell mRNA of a salmonid, the rainbow trout (Oncorhynchus mykiss). Trout DH segments are short and cannot be classified into independent DH families. Several of the ten identified putative DH segments had stretches of nucleotide sequence identity with mouse (DQ52, DFL 16.2 and Dsp 2.1), human (DM1) and chicken (DH4) DH. There was a clear preference for one or two of the three putative DH reading frames and a stop codon is often present in the less used reading frame. Four of the six JH segments are preferentially used, and analysis of the VH-DH and DH-JH junctions suggest the presence of N-nucleotides. The absolute size and size heterogeneity of the rainbow trout CDRH3 are smaller than those of the Xenopus, mouse and human CDRH3. About 75% of the 84 in-frame trout CDRH3 have 8, 9 or 10 residues and none of them have more than 11 residues. This homogeneization of the CDRH3 loop size may partly explain the restricted antibody diversity in lower vertebrates.

Amino Acid Sequence↗
PubMed1997

Microsites for immunoglobulin switch recombination breakpoints from Xenopus to mammals.

Immunoglobulin (Ig) heavy chain class switch recombination has been studied at the DNA level in a non-mammalian vertebrate, the amphibian Xenopus. A switch (S) region of about 5 kb has been identified in the JH-C mu intron of the Ig heavy chain locus in Xenopus. S mu contains 23 repeats approximately 150 bp long. Each repeat consists of internal shorter repeats and palindromic sequences, such as AGCT, which they share with mammalian switch regions. A deletion of the mu gene and the joining of the S regions of mu and chi occurs in B cells expressing IgX, one of the two non-mu isotypes in Xenopus. S chi shows no sequence homology with S mu and is characterized by 16 and 121 bp repeats and a high frequency of CATG, AGCA and TGCA palindromes. Both IgM and IgX S regions are AT rich and not GC rich like mammalian S regions. Recombination occurs, most of the time, at positions (microsites) where a single-stranded DNA folding program predicts the transition from a stem to a loop structure. This feature is conserved in most mammalian switch junctions which points to the general existence and involvement of microsites at one step of the determination of the recombination break-point. The recombinogenic nature of the switch regions is therefore linked to its structure rather than to its base composition, the repetitive occurrence of palindromes being essential at creating many microsites.

Animals↗
PubMed2004

A novel gene, FGA7, is fused to RUNX1/AML1 in a t(4;21)(q28;q22) in a patient with T-cell acute lymphoblastic leukemia.

AML1 is among the most frequent targets of chromosomal rearrangements in human leukemias. We report here the molecular analysis of a t(4;21)(q28;q22) that has disrupted AML1 in a patient with de novo T-cell acute lymphoblastic leukemia. By using 3'-RACE analysis, we show that this rearrangement results in the fusion of a novel gene immediately downstream of exon 5 or exon 6 of AML1, indicating that the AML1 breakpoint lies in intron 6 and that alternative fusion splice variants are generated. The sequence of the novel gene, located at 4q28, does not have any significant homology with any of the known genes in the human GenBank DNA database. However, the first 118 bases are identical to a part of a human ovarian EST. Also, its high homology with mouse and rat sequences suggests that this sequence most probably represents a part of a novel gene, which we named FGA7 (Fused Gene 7 to AML1). Following the AML1 open reading frame, the FGA7 sequence encodes an unknown protein of 27 amino acids. We isolated three bacterial artificial chromosome (BAC) clones that contain the FGA7 sequence and confirmed the breakpoint of the gene on the patient's metaphase spreads by fluorescence in situ hybridization using these BACs as probes. RT-PCR and Northern blot analyses revealed that FGA7 is expressed in ovarian and skeletal muscle tissues. The predicted AML1-FGA7 chimeric proteins contained a limited number of residues fused to AML1 in a situation similar to that reported for the AML1-EAP fusion that is a product of t(3;21). It is possible that the expression of a constitutively shortened AML1 could compete with full-length AML1 and act as a dominant negative inhibitor of the promoters that the core binding factor activates.

Amino Acid Sequence↗