Isolation and characterization of a cDNA encoding the zebra finch myelin proteolipid protein

A cDNA was isolated from a zebra finch telencephalon cDNA library that encodes the myelin proteolipid protein. The clone was 2874 nucleotides long containing an open reading frame of 831 nucleotides that encoded a 277 amino acid myelin proteolipid protein. The 5'- and 3' untranslated regions were 112 and 1931 nucleotides, respectively. In Northern blots the clone hybridized to 3 bands of 3.5, 2.4 and 1.5 Kb in mouse brain RNA, but to only a single band of 3.0 kb in zebra finch brain RNA, suggesting the lack of alternative polyadenylation sites within the 3' untranslated region of the zebra finch PLP mRNAs. There was a small degree of homology between the zebra finch and chicken PLP 5' untranslated regions, but relatively little homology of the 5' untranslated regions of the zebra finch PLP cDNA clone with the homologous regions of PLP cDNAs of many mammalian species. Except for a small stretch of considerable homology, there was little overall homology with the 3' untranslated regions of mammalian PLP mRNAs. Approximately 10% (i.e. 28) of the amino acids in the zebra finch PLP differed from mammalian PLP, with most of these changes located within exon 3. There were 16 amino acid changes between zebra finch and chicken, suggesting that greater sequence variation in PLP structure is tolerated among avian species than among mammalian species.     

Redesigning primary care processes to improve the offering of mammography. The use of clinic protocols by nonphysicians

We have isolated a genomic clone corresponding to a caffeic acid O-methyltransferase (COMT) from barley (Hordeum vulgare L.) using a cDNA for a previously described jasmonate-regulated mRNA showing homology to COMT. Primer extension was used to characterize the 5' end of the mRNA while the 3' end, intron/exon structure and other features of the sequence were deduced by comparison to the cDNA sequence and/or conserved motifs. The gene is mapped to chromosome five and is absent in the barley cultivar Morex. Southern and northern analyses suggest that no differences in genomic structure and jasmonate inducibility exist between the barley cultivar Salome (source of the cDNA clone) and Igri (source of the genomic clone). This genomic clone is thus suitable for promoter studies with respect to jasmonate induction.     

Benign monoclonal gammopathies

A cDNA clone encoding bone morphogenetic protein 4 (BMP 4) has been isolated from a primary fetal rat calvarial cell cDNA library. Sequencing of this clone has revealed a single open reading frame encoding a 408 amino acid protein. Comparison of 5' noncoding exon 1 portion of this cDNA with that of human bone and prostate BMP4 cDNA shows that BMP4 gene expression may possess tissue specificity.     

Isolation and characterization of a 2.3-kilobase-pair cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor

An immunoscreening strategy was used to isolate a cDNA clone encoding the binding domain for the external glycoprotein gp51 of the bovine leukemia virus (BLV). Three recombinant phages demonstrating BLV binding activity and containing 2.3-kbp cDNA inserts with identical nucleotide sequences were isolated from a lambda gt11 cDNA library of bovine kidney cells (MDBK). One clone, BLVRcp1, hybridized with a 4.8-kb mRNA from cells of bovine origin and was also found to be conserved as a single-copy gene in murine, bovine, ovine, primate, canine, feline, and porcine DNAs. The same gene is amplified in caprine DNA isolated from a BLV-induced tumor. The longest open reading frame of BLVRcp1 encodes a protein fragment of 729 amino acids with a putative receptor structure. BLVRcp1 cDNA was cloned in the eucaryotic expression vector pXT-1 and transfected into murine NIH 3T3 and human HEp-2 cells. Cells expressing BLVRcp1 mRNA became susceptible to BLV infection. BLVRcp1 has no known physiological function and has no significant homology with sequences registered in the GenBank and EMBL data libraries (31 July 1992). Expression of deleted constructs of BLVRcp1 indicates that the BLV binding region is encoded at the 5' side of the receptor clone.     

Molecular cloning, heterologous expression, and characterization of human glyoxalase II

A clone encoding glyoxalase II has been isolated from a human adult liver cDNA library. The sequence of 1011 base pairs consists of a full-length coding region of 780 base pairs, corresponding to a protein with a calculated molecular mass of 28,861 daltons. Identities (50-60%) were found to partial 5' and 3' cDNA sequences from Arabidopsis thaliana as well as within a limited region of glutathione transferase I cDNA from corn. A vector was constructed for heterologous expression of glyoxalase II in Escherichia coli. For optimal yield of enzyme, silent random mutations were introduced in the 5' coding region of the cDNA. A yield of 25 mg of glyoxalase II per liter of culture medium was obtained after affinity purification with immobilized glutathione. The recombinant enzyme had full catalytic activity and kinetic parameters indistinguishable from those of the native enzyme purified from human erythrocytes.     

Cloning and characterization of a developmentally regulated sea urchin cDNA encoding glutamine synthetase

A 2935-bp cDNA clone encoding glutamine synthetase (GS) was isolated from a cDNA library prepared from four-blastomere Paracentrotus lividus sea urchin embryos. The sequence consists of a 75-bp 5' untranslated region (5'-UTR) followed by a 1095-bp coding region corresponding to a 365-amino-acid (aa) protein, a 1747-bp 3'-UTR and a terminal 18-bp poly(A) tail. The encoded protein shows about 66% identical residues, as compared with human and lobster class-II GS. The sequence contains the Mn(2+)-binding aa and the highly conserved aa regions observed in other GS. Northern blot analyses show that the GS mRNA is present in the sea urchin egg and is developmentally regulated in the embryo.     

Cloning and characterization of a Schistosoma mansoni cDNA clone with a specific antigenic expression during development in a vertebrate host

Approximately 2.0 x 10 cDNA clones of an Schistosoma mansoni lambda gt11 cDNA library were screened in duplicate with serum from infected mice corresponding to distinct phases of infection. A cDNA clone (7/1) was isolated and recognized only by seven week serum. The clone was subcloned in pGEX-2T and Western-blot studies showed a specific antigenic expression confirming that only serum from the chronic phase is capable of recognizing this antigen. Dot-hybridization with RNA from different developmental phases of the parasite showed that the corresponding 7/1 RNA is expressed in all phases of parasite development in vertebrate hosts.     

Identification of a cDNA encoding 6-phosphogluconate dehydrogenase from a human heart cDNA library

A full-length cDNA clone for human 6-phosphogluconate dehydrogenase (PGD) was isolated from a human adult heart cDNA library. The clone encoded an open reading frame of 483 amino acids. When the amino acid sequences of human PGD and sheep PGD were aligned, 94.2% identity between these two proteins was found. Its calculated molecular weight is 53,149 daltons. The predicted isoelectric point is 6.85. When the secondary structure of human PGD was examined by the PROSIS software, 36% alpha-helix and 9% beta-sheet were found.     

The abundant 31-kilodalton banana pulp protein is homologous to class-III acidic chitinases

We have identified and characterized the abundant protein from the pulp of banana fruit (Musa acuminata cv. Grand Nain), and have isolated a cDNA clone encoding this protein. Comparison of the amino terminal sequence of the purified 31 kDa protein (P31) suggests that it is related to plant chitinases. Western analyses utilizing rabbit anti-P31 antiserum demonstrate that this protein is pulp-specific in banana. A full-length cDNA clone homologous to class III acidic chitinase genes has been isolated from a pulp cDNA library by differential screening. The identity of this clone as encoding P31 was verified by comparisons between the amino-terminal peptide sequence and the cDNA sequence and cross-hybridization of the translation product of the cDNA clone with P31 antiserum. Northern and western blot analyses of RNA and protein isolated from banana pulp at different stages of ripening indicate that the cDNA and protein are expressed at high levels in the pulp of unripe fruit, and that their abundance decreases as the fruit ripens. Based on its expression pattern and deduced amino acid sequence and composition, we hypothesize that the physiological role of P31 is not for plant protection, but as a storage protein in banana pulp.     

Characterization of the mRNA encoding a proline-rich 37-kilodalton glycoprotein from the excretory-secretory products of Trichostrongylus colubriformis

A glycoprotein, with apparent molecular weight in SDS-polyacrylamide gels of 37 kDa, has been isolated from the excretory-secretory (ES) products of the adult stage of Trichostrongylus colubriformis, a parasitic nematode. This protein is the major ES product recognized in immunoblots by lymph from a naturally infected sheep. A synthetic oligonucleotide, based on peptide sequence data from a digest of the purified protein was used to successfully screen a cDNA library. A cDNA clone was isolated which encoded a presumptive protein precursor of 220 amino acids that contained a 63 amino acid region of which more than 35% of the residues were proline, three peptide sequences determined from the natural component, and three potential N-glycosylation sites, consistent with the protein being isolated from the lectin-bound fraction of the adult ES products. The presumptive, processed, amino terminus encoded by the cDNA clone was preceded by a signal-like, hydrophobic-rich region of 16 amino acids.     

Advantage of polymerase chain reaction in the diagnosis of herpes simplex encephalitis: presentation of 5 atypical cases

Exon trapping from cosmids mapping to chromosome 19q13.3 yielded 6 exonic sequences that matched the human symplekin gene, which encodes a tight junction-related protein. One exonic sequence identified a 4.0 kb brain cDNA clone, R6E1, which contained 302 bp 5' to the originally reported 3.7 kb symplekin cDNA. A portion of this novel 5' sequence matched an additional trapped exonic sequence which was obtained from the most telomeric cosmid analyzed. The symplekin gene thus lies in a telomeric-to-centromeric direction on 19q13.3. Only three cosmids from a large 19q13.3 contig hybridized with R6E1, thereby assigning the symplekin gene to a 40 kb region immediately telomeric to gene 59 and the DM protein kinase gene. The 5' end of the R6E1 clone has a potential initiation codon with a strong Kozak sequence and Northern blot analysis detected a 4.2 kb signal in most human tissues, indicating that R6E1 may be a complete cDNA sequence. Based on the trapped exonic sequences, twelve exon-intron boundaries were predicted.     

Cloning of a human cDNA for CTP-phosphoethanolamine cytidylyltransferase by complementation in vivo of a yeast mutant

CTP-phosphoethanolamine cytidylyltransferase (ET) is the enzyme that catalyzes the formation of CDP-ethanolamine in the phosphatidylethanolamine biosynthetic pathway from ethanolamine. We constructed a Saccharomyces cerevisiae mutant of which the ECT1 gene, putatively encoding ET, was disrupted. This mutant showed a growth defect on ethanolamine-containing medium and a decrease of ET activity. A cDNA clone was isolated from a human glioblastoma cDNA expression library by complementation of the yeast mutant. Introduction of this cDNA into the yeast mutant clearly restored the formation of CDP-ethanolamine and phosphatidylethanolamine in cells. ET activity in transformants was higher than that in wild-type cells. The deduced protein sequence exhibited homology with the yeast, rat, and human CTP-phosphocholine cytidylyltransferases, as well as yeast ET. The cDNA gene product was expressed as a fusion with glutathione S-transferase in Escherichia coli and shown to have ET activity. These results clearly indicate that the cDNA obtained here encodes human ET.