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At least 1,063 records · Page 59Linked to original sources
PubMed1994

Expression of synthetic cDNA sequences encoding human insulin-like growth factor-1 (IGF-1) in the mammary gland of transgenic rabbits.

We have developed an expression system where foreign proteins are synthesized specifically in the mammary gland of transgenic rabbits and secreted into the milk. Regulatory elements were isolated from the bovine alpha S1-casein-encoding gene and combined with a synthetic DNA coding for human IGF-1 and for [Gln58]IGF-1, an IGF-1 analogue. The resulting hybrid DNA constructs were used to generate transgenic rabbits. Females of seven transgenic lines tested were positive for synthesis of IGF-1. Transmission of the transgene to progeny and IGF-1 production in female offspring was observed in all transgenic lines analysed. As expected, expression of transgene mRNA could only be detected in the mammary gland. Production levels of transgenic protein were as high as 1 g IGF-1 per liter rabbit milk. IGF-1, as well as [Gln58]IGF-1, when secreted into rabbit milk, was correctly processed and biologically active. IGF-1 was purified from the milk of transgenic rabbits to a nearly homogenous active form.

Animals↗
PubMed1991

cDNA sequence and genomic structure of EV12B, a gene lying within an intron of the neurofibromatosis type 1 gene.

The gene responsible for neurofibromatosis type 1 (NF1), one of the more common inherited human disorders, was identified recently, and segments of it were cloned. Two translocation breakpoints that interrupt the NF1 gene in NF1 patients flank a 60-kb segment of DNA that contains the EV12A locus (previously reported as the EV12 locus), the human homolog of a mouse gene, Evi-2A, implicated in retrovirus-induced murine myeloid tumors. EVI2A lies within an intron of the NF1 gene and is transcribed from telomere toward centromere, opposite to the direction of transcription of the NF1 gene. Here we describe a second locus, EVI2B, also located between the two NF1 translocation breakpoints. Full-length cDNAs from the EV12B locus detect a 2.1-kb transcript in bone marrow, peripheral blood mononuclear cells, and fibroblasts. Sequencing studies predict an EV12B protein of 448 amino acids that is proline-rich and contains an N-terminal signal peptide, an extracellular domain with four potential glycosylation sites, a single hydrophobic transmembrane domain, and a cytoplasmic hydrophilic domain. At the level of genomic DNA the EV12B locus lies within the same intron of the NF1 gene as EV12A and contains a 57-bp 5' exon that is noncoding, an 8-kb intron, and a 2078-bp 3' exon that includes the entire open reading frame. EV12B is transcribed in the same direction as EV12A; its 5' exon lies only 4 kb downstream from the 3' exon of the EV12A locus. In the mouse the 5' exon of the homologous gene, Evi-2B, lies approximately 2.8 kb from the 3' end of Evi-2A, in the midst of a cluster of viral integration sites identified in retrovirus-induced myeloid tumors; thus, Evi-2B may function as an oncogene in these tumors.

Amino Acid Sequence↗
PubMed2004

Characterization of the bovine neurofilament NF-M protein and cDNA sequence, and identification of in vitro and in vivo calpain cleavage sites.

Neurofilaments are the major components of the neuronal cytoskeleton, and accumulations of these proteins are associated with several important human diseases. Here we report the cloning and sequencing of bovine NF-M, the first NF-M cloned from a large domestic mammal. The bovine sequence proves to be generally more similar to that of human NF-M than the previously described mammalian sequences, suggesting that bovine neurofilaments are a useful model for biochemical studies of application to humans. However, we noted some unusual features within the 16 lys-ser-pro (KSP2) type phosphopeptide repeats and also note that the number of these repeats correlates well with the size of animal. We also characterized two in vitro calpain cleavage sites by direct peptide sequencing, finding that both are located in the glutamic acid rich E segment. Finally, we show biochemically that the more abundant and stable of these calpain fragments can also be detected in vivo.

Amino Acid Sequence↗
PubMed2006

Cloned Vero cell lines transfected with full-length A-segment or ORF1 cDNA sequence of IBDV.

Recombinant plasmids containing the A-segment or VP2/4/3 gene of infectious bursal disease virus (IBDV) were transfected into Vero cells. Monoclonal Vero cell lines were generated under G418 selection. Genomic (PCR/Southern blot) and transcriptional (RT-PCR/Northern blot) analyses showed that one copy of A-segment or VP2/4/3 or a partial gene of them was randomly and stably inserted into genomic DNA of Vero cells, and was able to transcribe corresponding mRNA. IFA/IPMA and Western blot analyses further confirmed that two of the monoclonal Vero cells with insertion of the A-segment of IBDV into genomic DNA could stably express VP2, VP3 and VP5 proteins, one cell line only expressed VP2 protein, and three monoclonal Vero cell lines with genomic insertion of the VP2/4/3 gene of IBDV could express VP2, VP3 and VP4 proteins. Under G418 selection, integrated foreign genes can be inherited along with cellular genomic DNA during cell replications. Moreover, DNA fragmentation and caspase-3 activity assays illustrated that cell apoptosis did not develop in monoclonal Vero cell lines expressing VP2 and VP5 proteins. The monoclonal IBDV gene-inserted Vero cell lines developed in this study will facilitate better understanding of IBDV and other members of the Birnaviridae in an expression system that would enable investigation of virus-host cell interactions on the cellular and molecular level.

Animals↗
PubMed2006

Identification of Heterodera glycines (soybean cyst nematode [SCN]) cDNA sequences with high identity to those of Caenorhabditis elegans having lethal mutant or RNAi phenotypes.

The soybean cyst nematode (SCN; Heterodera glycines) is a devastating obligate parasite of Glycine max (soybean) causing one billion dollars in losses to the US economy per year and over ten billion dollars in losses worldwide. While much is understood about the pathology of H. glycines, its genome sequence is not well characterized or fully sequenced. We sought to create bioinformatic tools to mine the H. glycines nucleotide database. One way is to use a comparative genomics approach by anchoring our analysis with an organism, like the free-living nematode Caenorhabditis elegans. Unlike H. glycines, the C. elegans genome is fully sequenced and is well characterized with a number of lethal genes identified through experimental methods. We compared an EST database of H. glycines with the C. elegans genome. Our goal was identifying genes that may be essential for H. glycines survival and would serve as an automated pipeline for RNAi studies to both study and control H. glycines. Our analysis yielded a total of nearly 8334 conserved genes between H. glycines and C. elegans. Of these, 1508 have lethal phenotypes/phenocopies in C. elegans. RNAi of a conserved ribosomal gene from H. glycines (Hg-rps-23) yielded dead and dying worms as shown by positive Sytox fluorescence. Endogenous Hg-rps-23 exhibited typical RNA silencing as shown by RT-PCR. However, an unrelated gene Hg-unc-87 did not exhibit RNA silencing in the Hg-rps-23 dsRNA-treated worms, demonstrating the specificity of the silencing.

Animals↗
PubMed1997

The cDNA sequences of cytochrome c oxidase subunit VIa from carp and rainbow trout suggest the absence of isoforms in fishes.

The cDNAs of subunit VIa of cytochrome c oxidase from rainbow trout liver and carp heart are presented, revealing 82% identity of their deduced amino acid sequences. The two cDNAs are evolutionary equally distant from the livertype (VIaL) and heart-type (VIaH) of mammalian subunit VIa. The data suggest that in ectotherm fishes no isoforms of subunit VIa occur, and that the postulated tissue-specific mechanism of thermogenesis in mammals, based on interaction of ATP with subunit VIaH (Frank, V. and Kadenbach, B. (1996) FEBS Lett. 382, 121-124), is absent.

Amino Acid Sequence↗
PubMed1997

The Arabidopsis thaliana cDNA sequencing projects.

Nearly 30000 Arabidopsis thaliana EST (Expressed Sequence Tags) have been produced by a French-American consortium. Despite redundancy, these sequences tag about half of the expected Arabidopsis genes. Approximately 40% of the non-redundant EST can be assigned a putative function by simple homology search. This programme allowed the identification of a large number of genes which would have been very difficult to isolate by other classical techniques. It considerably stimulated many areas of plant biology by the rapid discovery a large number of genes, by revealing multigene families and by allowing the analysis of differential expression of the different members. Finally this programme facilitated construction of physical maps of the chromosomes and opened the way for complete sequencing of the Arabidopsis genome and comparative mapping of the major plant crops.

Arabidopsis↗
PubMed1997

The Arabidopsis thaliana cDNA sequencing projects.

Nearly 30000 Arabidopsis thaliana EST (Expressed Sequence Tags) have been produced by a French and an American consortium. Despite redundancy these sequences tag about half the expected Arabidopsis genes. Approximately 40% of the non-redundant EST can be assigned a putative function by simple homology search. This programme allowed the identification of a large number of genes which would have been very difficult to isolate by other classical techniques. It considerably stimulated many areas of plant biology by the rapid discovery a large number of genes, by revealing multigene families and by allowing the analysis of differential expression of the different members. Finally this programme facilitated construction of physical maps of the chromosomes and opened the way for complete sequencing of the Arabidopsis genome and comparative mapping of the major plant crops.

Arabidopsis↗
PubMed1998

cDNA sequence analysis and expression of four long neurotoxin homologues from Naja naja atra.

The novel cDNAs encoding four long neurotoxin homologues were firstly cloned from the venom gland of Naja naja atra by reverse transcriptase-polymerase chain reaction. Amino acid sequence comparison showed that they may come from two different evolutionary origins. The mature proteins were expressed as soluble fusion proteins in Escherichia coli and purified for immunoblotting. The results revealed that they displayed different immunochemical properties.

Amino Acid Sequence↗
PubMed1998

The mouse Fkh1/Mf1 gene: cDNA sequence, chromosomal localization and expression in adult tissues.

The 'winged helix' or 'forkhead' transcription factor gene family is defined by a common 100-amino-acid DNA-binding motif. Here, we describe the chromosomal position, start site of transcription, sequence and adult expression pattern of the mouse Fkh1/Mf1 (Forkhead homologue 1/mesoderm/mesenchyme forkhead 1) gene. This gene contains one exon and encodes a protein of 553 amino acids that is highly related to the mouse MFH1 protein. The Fkh1/Mf1 mRNA is expressed widely in adult tissues. Linkage analysis showed that the Fkh1/Mf1 gene is localized to chromosome 13 at 17.02cM from the centromer, in close proximity to Bmp6 and Hfh1, another distinct member of the winged helix gene family.

Amino Acid Sequence↗
PubMed1997

cDNA sequence and expression pattern of the Drosophila melanogaster PAPS synthetase gene: a new salivary gland marker.

PAPS synthetase is a bifunctional enzyme containing both ATP sulfurylase and APS kinase activities required for the biosynthesis of PAPS, the sulfate donor in sulfation reactions. Here we report the sequence of the Drosophila melanogaster PAPS synthetase, the first gene implicated in the sulfation pathway to be described in that organism, and the characterization of its specificity of expression in embryos. Whole-mount in situ hybridization reveals that DmPAPSS is a novel salivary gland marker. At the end of embryogenesis, expression of DmPAPSS is also observed at the entry and exit of the gut and the posterior spiracles. We discuss the possibility that the pattern of expression of the DmPAPSS gene might reflect a major role for sulfation in mucus biosynthesis at the end of Drosophila embryogenesis.

Amino Acid Sequence↗
PubMed1997

cDNA sequencing of the 5' noncoding region (5' NCR) to determine hepatitis C genotypes in patients with chronic hepatitis C.

Reports suggest that response to interferon-alpha therapy is influenced by both hepatitis C viral genotype and titer. Our aim was to determine if direct, automated, cycle sequencing of the PCR product from an HCV RNA detection assay could be used to reliably determine HCV genotype. In addition, the approach was used to determine the HCV genotype distribution in our patient population and to learn if there was a correlation between HCV genotype and RNA titer that could be used to predict response to treatment. In all 143 consecutive patients were tested for both HCV RNA titer and genotype. Automated, cycle sequencing of PCR product was highly effective and failed to yield a genotype in only 3 (2%) patients. The distribution of HCV genotypes was: 1a (40%), 1b (39%), 2a (2%), 2b (6%), 3a (4%). There were significant differences in the median HCV RNA titers between genotypes 1, 2, and 3. High HCV RNA titers >4.4 x 10(6) copies/ml were only seen in genotype 1. However, the HCV RNA level should not be used as a surrogate marker of genotype because of a significant overlap of titers within the genotypes.

Base Sequence↗
PubMed2003

The entire cDNA sequences of projectin isoforms of crayfish claw closer and flexor muscles and their localization.

Projectin is a giant protein related to twitchin and titin/connectin, that is found in arthropod striated muscle. The complete sequence of a 1 MDa projectin from Drosophila muscle was recently deduced from a thorough analysis of the genomic DNA (Southgate and Ayme-Southgate, 2001). Here we report the complete sequence for projectin from crayfish claw closer muscle (8625 residues; 962,634 Da). The N-terminal sequence contains 12 unique 19-residue repeats rich in glutamic acid (E) and lysine (K). This region, termed the EK region, is clearly distinguishable from the PEVK-like domain of Drosophila projectin. The sequence of crayfish flexor projectin differs from that of closer muscle projectin in that there is a 114-residue deletion and a 35-residue insertion in the N-terminal region. Immunofluorescence microscopy demonstrated that projectin is mainly localized within the sarcomeric A band in both closer and flexor muscles, although the N-terminal region was shown to extrude into the I band region. In the closer muscles, invertebrate connectin (D-titin) connects the Z line to the edge of the A band (Fukuzawa et al., 2001). We have shown that invertebrate connectin is also present in flexor muscle sarcomeres, although in very low abundance.

Amino Acid Motifs↗
PubMed2003

Identification of differentially expressed cDNA sequences in ovaries of sexual and apomictic plants of Brachiaria brizantha.

The isolation of genes associated with apomixis would improve understanding of the molecular mechanism of this mode of reproduction in plants as well as open the possibility of transfer of apomixis to sexual plants, enabling cloning of crops through seeds. Brachiaria brizantha is a highly apomictic grass species with 274 tetraploid apomicts accessions and only one diploid sexual. In this study we have compared gene expression in ovaries at megasporogenesis and megagametogenesis of sexual and apomictic accessions of B. brizantha by differential display (DD-PCR), with 60 primer combinations. Specificity of 65 cloned fragments, checked by reverse northern blot analysis, showed that 11 clones were differentially expressed, 6 in apomictic ovaries, 2 in sexual and 3 in apomictic and sexual, but at different stages. Of the 6 sequences isolated that were preferentially expressed in the apomictic accession: one sequence was from ovaries at megasporogenesis stage; three were from megagametogenesis stage; two were from both stages. Of the two sequences isolated from the sexual accessions, one showed expression in ovaries at megagametogenesis, while the other sequence was shown to be specific to both stages. Three sequences were from megasporogenesis stage in apomicts but were also detected at megagametogenesis in sexual plants. Sequence analysis showed that 5 of the 11 clones had no apparent homologues in the protein database. Some of the clones identified as apomictic-specific shared homology with known genes enabling their functional annotation. The relationships of these functions to the generation of the apomictic trait are discussed.

Amino Acid Sequence↗
PubMed1981

Molecular cloning and characterization of cDNA sequences coding for rat relaxin.

Using a synthetic oligonucleotide primer, cloned DNA fragments, each containing the entire coding sequence of rat relaxin, have been isolated from a clone 'bank' of ovarian mRNA sequences. The nucleotide sequence of these clones demonstrates that relaxin is synthesized as a preprorelaxin molecule with an unexpectedly large connecting peptide of 105 amino acid residues.

Amino Acid Sequence↗
PubMed1981

Homology between the primary structure of alpha-fetoprotein, deduced from a complete cDNA sequence, and serum albumin.

The entire DNA complementary to murine alpha-fetoprotein mRNA has been cloned in the plasmid vector pBR322 and its complete nucleotide sequence determined. The deduced amino acid sequence identifies a hydrophobic prepeptide of 19 amino acids and the complete primary structure of alpha-fetoprotein. Its structure reveals extensive homology to serum albumin and suggests that there are 15 disulphide bridges in the alpha-fetoprotein molecule, all at the same positions as those found in albumin. The AFP molecule can be organized into a three-domain structure almost identical to that of serum albumin, suggesting a common ancestral origin of the two proteins.

Amino Acid Sequence↗