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PubMed1988

[Colony-stimulating factors in cancer chemo- and radiotherapy].

Recombinant human granulocyte colony-stimulating factor (Re Hu G-CSF) was prepared and its stimulating effect on granulocytopoiesis was examined in mice. Human G-CSF was purified to homogeneity from conditioned media of a G-CSF-producing cell line. The amino-terminal sequence was determined. By using oligonucleotides as probes, determined by the amino acid sequence, a cDNA library prepared from human macrophages was screened. The cloned G-CSF cDNA was expressed in E. coli K12MM294, and the mature protein was purified to homogeneity. Mice were given intraperitoneal injections of Re Hu G-CSF every day for 14 days. Peripheral blood granulocyte counts were examined 4, 8, 12 and 14 days after injection. Mice were sacrificed on the 14th day for histologic examinations of the bone marrow and spleen. Granulocyte counts began to increase on the 4th day and reached about 80,000/mm3 on the 14th day. Cells of granulocyte lineage were markedly increased in the bone marrow and spleen. Granulocyte precursors (CFU-C) were remarkably increased in the spleen. When mice were treated with 5-fluorouracil, cyclophosphamide or irradiation, the period of granulocytopenia was significantly shortened by subcutaneous injection of Re Hu G-CSF. These results suggest that human G-CSF play a central role in granulocyte production in vivo. The ability of Re Hu G-CSF to stimulate granulocyte production implies that this factor will be clinically useful in neutropenic patients treated with anti-cancer agents or irradiation.

Agranulocytosis↗
PubMed1997

Podoplanin, novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis.

Puromycin aminonucleoside nephrosis (PAN), a rat model of human minimal change nephropathy, is characterized by extensive flattening of glomerular epithelial cell (podocyte) foot processes and by severe proteinuria. For comparison of expression of glomerular membrane proteins of normal and PAN rats, a membrane protein fraction of isolated rat glomeruli was prepared and monoclonal antibodies were raised against it. An IgG-secreting clone designated LF3 was selected that specifically immunolabeled podocytes of normal but not of PAN rats. The antigen of LF3 IgG was identified as a 43-kd glycoprotein. Molecular cloning of its cDNA was performed in a delta gt11 expression library prepared from mRNA of isolated rat glomeruli. The predicted amino acid sequence indicated a 166-amino-acid integral membrane protein with a single membrane-spanning domain, two potential phosphorylation sites in its short cytoplasmic tail, and six potential O-glycosylation sites in the large ectodomain. High amino acid sequence identities were found to membrane glycoproteins of rat lung and bone and mouse thymus epithelial cells as well as to a phorbol-ester-induced protein in a mouse osteoblast cell line and to a canine influenza C virus receptor. In PAN, expression of this 43-kd protein was selectively reduced to < 30%, as determined by quantitative immunogold electron microscopy, immunoblotting, and Northern blotting. These data provide evidence that transcription of the 43-kd transmembrane podocyte glycoprotein is specifically down-regulated in PAN. To indicate that this protein could be associated with transformation of arborized foot processes to flat feet (Latin, pes planus) we have called it podoplanin.

Amino Acid Sequence↗
PubMed1994

In-tube cDNA cloning method using a biotinylated RNA probe.

We report a simple, practical method for isolating a particular cDNA from a single-stranded (ss) cDNA library in a tube without using a radioisotope. The method consists of three steps: (i) the capture of a target cDNA by a biotinylated RNA via intermolecular hybrid formation, (ii) the binding of the cDNA-RNA hybrid to an avidin-coated gel, (iii) the recovery of the target cDNA by degradation of the RNA under mild alkaline conditions. The effectiveness of the method was examined by isolating a clone carrying a metapyrocatechase gene from a model library. This model experiment achieved an enrichment of 4800-fold. This method was applied to cloning of a cDNA encoding interleukin 8 (IL-8) from a cDNA library prepared from phorbol myristate acetate-treated U937 cells. Using an RNA probe prepared from a truncated cDNA encoding IL-8, the corresponding full-length cDNA clones were successfully obtained.

Biotin↗
PubMed1989

Isolation and characterization of a maize chlorophyll a/b binding protein gene that produces high levels of mRNA in the dark.

A cDNA library prepared using mRNA isolated from red-light irradiated maize seedlings was screened by a difference procedure for clones that represent red-light regulated mRNA. Two such clones were found to represent mRNA for a chlorophyll a/b binding protein (CAB), and one of these (pAB1084) was used to screen a maize genomic library. One positive genomic clone (lambda AB1084) was isolated and sequenced. The gene represented by lambda AB1084, which we designate maize cab-1, contains extensive nucleotide homology within its protein coding region to CAB genes from other species. The boundaries of the transcribed region of the cab-1 gene were determined by S1 nuclease mapping. The 5' terminus of cab-1 mRNA is located 52-54 nucleotides (nt) upstream of the translation start site and 34 nt downstream of a TATA box. As in the case of petunia CAB genes, several poly(A) addition sites are present in mRNA from the cab-1 gene. The 5' flanking DNA of cab-1 contains sequences related to elements that have been implicated in the light-regulated expression of CAB and rbcS genes in other plant systems. Quantitative Northern blot hybridization analysis using a gene specific probe for cab-1 indicates that the mRNA for this gene is present at 0.4% of the total mRNA and up to 80% of the total CAB mRNA in the leaves of dark-grown seedlings. In consequence, although the degree of up-regulation by white light is only moderate (3- to 6-fold), cab-1 transcripts account for approximately 2% of the mRNA in the leaves of light-grown seedlings.

Amino Acid Sequence↗
PubMed1990

Cloning and expression of a cDNA encoding human placental protein 11, a putative serine protease with diagnostic significance as a tumor marker.

The placental protein 11 (PP11) can act as a tumor marker because of its specific association with various forms of cancer. A lambda gt11 cDNA library prepared from human placenta was screened with a polyclonal anti-PP11 antiserum. Out of 10(6) independent clones, only one clone reacted with the anti-PP11 antiserum. The isolated cDNA coded only for the carboxy-terminal part of PP11 and was subsequently used to rescreen a lambda gt10 placental cDNA library. Two cDNA clones out of 10(6) screened were identified encoding the entire protein of 369 amino acids, including a typical hydrophobic signal sequence of 18 amino acids. Expression of the PP11 cDNA coding sequence in Escherichia coli resulted in the synthesis of a protein with the expected size which can be specifically immunoprecipitated with anti-PP11 antiserum. Fractionation experiments revealed that two forms of the protein are present in the bacterial cell: a higher-molecular-weight form of approximately 42 kD in the cytoplasm and a smaller-molecular-weight form of approximately 42 kD in the periplasm. This result indicates that PP11 can be synthesized in E. coli and is process by removal of the hydrophobic signal sequence. Both the placental and the processed recombinant PP11 protein exhibit a protease activity.

Amino Acid Sequence↗
PubMed1996

Cloning and characterization of several cDNAs for UDP-glucose pyrophosphorylase from barley (Hordeum vulgare) tissues.

Eleven cDNA clones encoding UDP-glucose pyrophosphorylase (UGPase) have been isolated from cDNA libraries prepared from seed embryo, seed endosperm and leaves of barley (Hordeum vulgare L.). The sequences were identical, with the exception of positioning of the poly(A) tail; at least five clones with different polyadenylation sites were found. For a putative full-length cDNA [1775 nucleotides (nt) plus polyadenylation tail], isolated from an embryo cDNA library, an open reading frame of 1419 nt encodes a protein of 473 amino acids (aa) of 51.6 kDa. An alignment of the derived aa sequence with other UGPases has revealed high identity to UGPases from eukaryotic tissues, but not from bacteria. Within the aa sequence, no homology was found to a UDP-glucose-binding motif that has been postulated for a family of glucosyl transferases. The derived aa sequence of UGPase contains three putative N-glycosylation sites and has a highly conserved positioning of five Lys residues, previously shown to be critical for catalysis and substrate binding of potato tuber UGPase. A possible role for N-glycosylation in the intracellular targeting of UGPase is discussed.

Amino Acid Sequence↗
PubMed2004

Estimating and comparing the rates of gene discovery and expressed sequence tag (EST) frequencies in EST surveys.

MOTIVATION: Expressed sequence tag (EST) surveys are an efficient way to characterize large numbers of genes from an organism. The rate of gene discovery in an EST survey depends on the degree of redundancy of the cDNA libraries from which sequences are obtained. However, few statistical methods have been developed to assess and compare redundancies of various libraries from preliminary EST surveys. RESULTS: We consider statistics for the comparison of EST libraries based upon the frequencies with which genes occur in subsamples of reads. These measures are useful in determining which one of several libraries is more likely to yield new genes in future reads and what proportion of additional reads one might want to take from the libraries in order to be likely to obtain new genes. One approach is to compare single sample measures that have been successfully used in species estimation problems, such as coverage of a library, defined as the proportion of the library that is represented in the given sample of reads. Another single library measure is an estimate of the expected number of additional genes that will be found in a new sample of reads. We also propose statistics that jointly use data from all the libraries. Analogous formulas for coverage and the expected numbers of new genes are presented. These measures consider coverage in a single library based upon reads from all libraries and similarly, the expected numbers of new genes that will be discovered by taking reads from all libraries with fixed proportions. Together, the statistics presented provide useful comparative measures for the libraries that can be used to guide sampling from each of the libraries to maximize the rate of gene discovery. Finally, we present tests for whether genes are equally represented or expressed in a set of libraries. Binomial and chi2 tests are presented for gene-by-gene comparisons of expression. Overall tests of the equality of proportional representation are presented and multiple comparisons issues are addressed. These methods can be used to evaluate changes in gene expression reflected in the composition of EST libraries prepared from different tissue types or cells exposed to different environmental conditions. AVAILABILITY: Software will be made available at http://www.mathstat.dal.ca/~tsusko

Algorithms↗
PubMed1985

Isolation of recombinant cDNAs encoding chicken erythroid delta-aminolevulinate synthase.

We report the isolation of cDNA clones encoding delta-aminolevulinate synthase (ALA synthase; EC 2.3.1.37), the first enzyme in the heme biosynthetic pathway in animal cells. The gene was isolated from a chicken erythroid cDNA library prepared in the bacteriophage lambda fusion/expression vector gt11, using rabbit antibody raised against the relatively abundant chicken liver enzyme. The chicken liver and red cell ALA synthase isozymes share substantial crossreactivity to the antibody, thereby allowing isolation of the erythroid-specific gene by using the heterologous antibody in immune screening of the red cell cDNA library. Preliminary analysis documenting the tissue specificity of transcription indicates that the enzyme is encoded by a highly homologous set of messages, which appear to differ in size in various avian tissues. From analysis using strand-specific RNA probes, it appears that the different ALA synthase mRNAs detected may be transcribed from a family of genes that are closely related in nucleotide sequence and are each regulated in a developmentally specific manner.

5-Aminolevulinate Synthetase↗
PubMed2001

Exploring the DNA-binding specificities of zinc fingers with DNA microarrays.

A key step in the regulation of networks that control gene expression is the sequence-specific binding of transcription factors to their DNA recognition sites. A more complete understanding of these DNA-protein interactions will permit a more comprehensive and quantitative mapping of the regulatory pathways within cells, as well as a deeper understanding of the potential functions of individual genes regulated by newly identified DNA-binding sites. Here we describe a DNA microarray-based method to characterize sequence-specific DNA recognition by zinc-finger proteins. A phage display library, prepared by randomizing critical amino acid residues in the second of three fingers of the mouse Zif268 domain, provided a rich source of zinc-finger proteins with variant DNA-binding specificities. Microarrays containing all possible 3-bp binding sites for the variable zinc fingers permitted the quantitation of the binding site preferences of the entire library, pools of zinc fingers corresponding to different rounds of selection from this library, as well as individual Zif268 variants that were isolated from the library by using specific DNA sequences. The results demonstrate the feasibility of using DNA microarrays for genome-wide identification of putative transcription factor-binding sites.

Amino Acid Sequence↗
PubMed1990

Cloning and cDNA sequence of a bovine submaxillary gland mucin-like protein containing two distinct domains.

A lambda gt11 cDNA library prepared from bovine submaxillary gland mRNA was screened with polyclonal anti-apo-bovine submaxillary mucin antibodies with the aim of obtaining the deduced amino acid sequence of the mucin core protein. One of the positive clones had a 1.8 kilobase (kb) cDNA insert and coded for an incomplete protein. A 2.0-kb cDNA clone was isolated by rescreening the library with the 1.8-kb cDNA. Nucleotide sequencing of the full-length 2.0-kb cDNA revealed an open reading frame that coded for a 563-amino acid protein. A striking feature of the cloned protein is the skewed distribution of the amino acids, most notably that of the hydroxy amino acids and cysteine. The amino-terminal domain of 339 residues is very rich in threonine, serine, and glycine and poor in cysteine, aspartic acid, tyrosine, phenylalanine, and tryptophan. In contrast, the carboxyl-terminal domain of 224 residues is rich in cysteine, aspartic acid, tyrosine, lysine, and asparagine and relatively poor in threonine, serine, and glycine. A search of the protein data bank for homologies to the deduced amino acid sequence revealed statistically significant matches to several proteins, including the porcine submaxillary apomucin fragment. The cysteine-rich domain by itself was not statistically homologous with any of the registered polypeptide sequences. RNA blot analysis using DNA probes corresponding to the mucin-like and cysteine-rich regions detected a nearly identical pattern of transcripts, demonstrating that the characterized clones are not artifacts of cDNA library construction. The blots also showed the presence of polydisperse transcripts in bovine submaxillary gland but no detectable hybridization signals in liver or brain RNA.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of cDNA clones encoding cdc2 homologues from Oryza sativa: a functional homologue and cognate variants.

Using probes obtained by PCR amplification, we have isolated two cognate rice cDNAs (cdc2Os-1 and cdc2Os-2) encoding structural homologues of the cdc2+/CDC28 (cdc2) protein kinase from a cDNA library prepared from cultured rice cells. Comparison of the deduced amino acid sequences of cdc2Os-1 and cdc2Os-2 showed that they are 83% identical. They are 62% identical to CDC28 of Saccharomyces cerevisiae and much more similar to the yeast and mammalian p34cdc2 kinases than to rice R2, a cdc2-related kinase isolated previously by screening the same rice cDNA library with a different oligonucleotide probe. Southern blot analysis indicated that the three rice clones (cdc2Os-1, cdc2Os-2 and R2) are derived from distinct genes and are each found in a single copy per rice haploid genome. RNA blot analysis revealed that these genes are expressed in proliferating rice cells and in young rice seedlings. cdc2Os-1 could complement a temperature-sensitive yeast mutant of cdc28. However, despite the similarity in structure, both cdc2Os-2 and R2 were unable to complement the same mutant. Thus, the present results demonstrate the presence of structurally related, but functionally distinct cognates of the cdc2 cell cycle kinase in rice.

Amino Acid Sequence↗
PubMed1988

Characterization of cDNA encoding human placental anticoagulant protein (PP4): homology with the lipocortin family.

A cDNA library prepared from human placenta was screened for sequences encoding the placental protein 4 (PP4). PP4 is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade. Partial amino acid sequence information from PP4-derived cyanogen bromide fragments was used to design three oligonucleotide probes for screening the library. From 10(6)independent recombinants, 18 clones were identified that hybridized to all three probes. These 18 recombinants contained cDNA inserts encoding a protein of 320 amino acid residues. In addition to the PP4 cDNA we identified 9 other recombinants encoding a protein with considerable similarity (74%) TO PP4, which was termed PP4-X. PP4 and PP4-X belong to the lipocortin family, as judged by their homology to lipocortin I and calpactin I.

Amino Acid Sequence↗
PubMed1982

Isolation and characterization of the rat tryptophan oxygenase gene.

Tryptophan oxygenase (TO, EC 1.13.1.12) from rat liver is subject to glucocorticoid and developmental control. To study the mechanism of regulation, TO mRNA sequences and the chromosomal TO gene were cloned. From a cDNA library prepared from rat liver poly(A)+ RNA enriched for TO mRNA, a recombinant plasmid containing TO cDNA sequences was identified by translation of hybrid-selected RNA and immunoprecipitation with antibodies directed against TO. This cDNA clone hybridizes to a mRNA 2000 bases long that is inducible by dexamethasone. With this clone as probe we isolated from a bacteriophage lambda rat DNA library genomic clones which together span a region of 32 kilobase pairs (kb). Heteroduplex analysis revealed that the gene extends over 19 kb and is interrupted by at least 11 introns. To characterize the presumptive control region the DNA sequence around the 5' end of the TO gene was determined. S1 nuclease protection experiments revealed two separate start sites for TO mRNA transcription within this region.

Animals↗
PubMed1984

Molecular cloning and carboxyl-propeptide analysis of human type III procollagen.

Two human cDNA libraries prepared from normal fibroblast (GM3348) and rhabdomyosarcoma (CCL136) mRNAs were screened under cross hybridization conditions with a genomic fragment coding for exons 2 and 3 of the avian type III procollagen COOH-propeptide (Yamada, Y., Mudryj, M., Sullivan, M. and deCrombrugghe, B. (1983) J. Biol. Chem. 258, 2758-2761). One cDNA clone containing a 1.12 kb insert was isolated from the CCL136 library and later used to identify a GM3348 derived clone with a 2.4 kb insert. Comparison of the human and avian type III C-terminal propeptides revealed much more divergence in the first 54 amino acids following the terminal cysteine of the triple helical region than is present in the alpha 1(I) and alpha 2(I) procollagen chains of these species. Analysis of poly (A+) RNA from normal human fibroblast and tumor cell lines showed that they differed greatly in the relative amounts of alpha 1(I), alpha 2(I), and alpha 1(III) mRNAs. Furthermore, as we previously reported for the alpha 1(I) and alpha 2(I) transcripts, multiple mRNAs also hybridize to the cloned alpha 1(III) DNA.

Amino Acid Sequence↗
PubMed2001

Screening for novel cry genes by hybridization.

AIMS: To develop an efficient and sensitive method to facilitate the search for novel Cry toxins. METHODS AND RESULTS: The method uses a cocktail of cry gene sequences as a hybridization probe to screen Bacillus thuringiensis (Bt) strains and gene libraries prepared from them. Under the hybridization and washing conditions used, cross-hybridization between genes from different cry families was not observed. Probes containing either partial or complete cry gene sequences produced similar patterns when hybridized to genomic DNA of several Bt strains although the pattern produced by the probe composed of entire gene coding regions was somewhat more complex. CONCLUSION: As a tool for gene library screening, hybridization with a mixture of cry gene sequences is an efficient means of detecting clones containing a diverse range of cry genes in a single step. SIGNIFICANCE AND IMPACT OF THE STUDY: This technique greatly improves the ease and efficiency of novel toxin gene discovery compared to previous methods.

Bacillus thuringiensis↗
PubMed1982

Rat preprocarboxypeptidase A: cDNA sequence and preliminary characterization of the gene.

Rat carboxypeptidase A cDNA clones have been isolated from a cDNA library prepared from pancreatic mRNA. An almost complete mRNA sequence has been deduced that predicts a polypeptide having 78% amino acid sequence homology with bovine carboxypeptidase A. The amino acid sequence of the activation and signal peptides of the carboxypeptidase A precursor were inferred from the nucleotide sequence. The cDNA was used as a probe to identify DNA fragments containing carboxypeptidase A sequences in a bacteriophage lambda library of rat genomic DNA. Heteroduplexes revealed that the DNA coding sequence occupies 5.5 kilobases and is interrupted by nine intervening sequences. The nucleotide sequence of the 5' end of the gene and the adjacent flanking region provides information on the site of initiation of transcription and the putative control regions. There is no evident relationship between the localization of intervening sequences in the gene and functional/structural domains of the protein.

Animals↗
PubMed1991

Early occurrence of immunoglobulin isotype switching in human fetal liver.

A cDNA library prepared from a human fetal liver of the first trimester of gestation was screened with Ig C mu, C gamma, C kappa and C lambda probes. Ten heavy chain clones were isolated and characterized by restriction mapping and partial sequencing. The absence of Ig light chain clone and the presence of pre-B-specific lambda-like transcripts suggest that the immune compartment of this cDNA library was mostly derived from pre-B cells. Three transcripts of mu, gamma 2 and gamma 4 isotypes contained a V-D-J-C region with an open reading frame and used members of the VHIV, VHIII and VHI families, respectively. Seven clones were derived from sterile transcripts, one C mu and six C gamma. In addition to C mu exons, the sterile mu transcript contained the 5' flanking germline region. By contrast, the gamma sterile transcripts used a 5' sequence that was spliced from the I gamma 1 region onto the first C gamma 1 exon. In addition several of these transcripts were derived from alternative splicing. The simultaneous expression of both sterile and functional gamma transcripts suggests that the switch mechanism operates in normal fetal liver very early in ontogeny.

Antigenic Variation↗