Biomedical subjects

K S LaForge

Publications and source records attributed to K S LaForge.

25 records · Page 2Linked to original sources

PubMed1989

Structure of the canine pancreatic lipase gene.

Identification of three overlapping clones in a canine genomic lambda phage library allowed us to determine a detailed restriction enzyme map of the primary transcriptional unit of the pancreatic lipase gene (15.5 kilobase pairs) as well as 15 and 6 kilobase pairs of 5'- and 3'-flanking regions, respectively. DNA sequence analysis provided the primary structure of (a) 1,345 nucleotides (nt) of 5'-flanking sequence including CAAT and TATA boxes at positions -112 and -35, respectively, and a class 2 glucocorticoid receptor binding sequence at position -97, (b) 13,127 out of approximately 15,500 nt of the transcriptional unit which is organized into 13 exon sequences, and (c) 1,270 nt of 3'-flanking sequence. Exon 1 encodes the entire 5'-nontranslated mRNA sequence; exon 2, the ATG initiation codon and the hydrophobic portion of the signal peptide; and exon 6, Ser154 which shows homology to the active Ser152 in the porcine enzyme. Comparison of the amino acid sequences of human lipoprotein lipase, rat hepatic lipase, and Drosophila yolk proteins 1, 2, and 3 with canine pancreatic lipase shows that the central region of highest homology (encoded by exons 6-8 in the dog gene) contains four highly conserved subregions which may play a critical role in enzyme-substrate and protein-ligand binding for lipases and yolk proteins, respectively. Comparison of the sequences of 10 lipases from prokaryotes and eukaryotes identifies a 9-residue consensus sequence surrounding the active serine which includes the previously identified sequence Gly-X-Ser-X-Gly. The hydrophobic nature of this sequence in the 10 lipases contrasts with the hydrophilic nature of the corresponding sequences in serine proteases and thus defines an active site serine consensus sequence specific for lipases. An analysis of 5'- and 3'-flanking and intron 1-4 sequences in transient expression studies with AR4-2J and 266-6 cells was unable to reveal tissue-specific promoter or enhancer sequences.

Amino Acid Sequence↗

PubMed1986

Primary structures of canine pancreatic lipase and phospholipase A2 messenger RNAs.

cDNA clones coding for phospholipase A2 and lipase mRNA have been identified in a full-length cDNA library constructed from canine pancreatic poly (A) + mRNA. Phospholipase A2 mRNA contains 562 nucleotides and codes for a preproenzyme of 146 amino acids (Mr = 16,251) containing a 15 residue signal peptide (MetLysPheLeuValLeuAlaAlaLeuLeuThrValAlaAlaAla), a seven residue activation peptide (GluGlyGlyIleSerProArg), and a 124 residue mature enzyme, phospholipase A2 (79.2% homology with the porcine enzyme). The 5' nontranslated sequence contains a region where eight of nine bases show potential hybridization to the 3' end of 18S ribosomal RNA. Lipase mRNA contains 1,493 nucleotides and codes for a preenzyme with 467 amino acids (Mr = 51,489) which contains a 17 residue signal peptide (MetValSerIleTrpThrIleAlaLeuPheLeuLeuGlyAlaAlaLysAla) and a 450 residue mature enzyme, lipase (75.6% homology with porcine lipase). The 5' noncoding sequences for phospholipase A2 (28 bases) and lipase (34 bases) mRNAs both have an adenosine base three positions preceding the AUG initiation codon but otherwise demonstrate no homology.

Amino Acid Sequence↗

PubMed1985

Differential regulation of trypsinogen mRNA translation: full-length mRNA sequences encoding two oppositely charged trypsinogen isoenzymes in the dog pancreas.

In the absence of changes in functional mRNA levels, stimulation of the pancreas with caerulein, a peptide analog of cholecystokinin, has been previously shown to increase the synthesis of anionic but not cationic trypsinogen. To look for structure-function correlations, a high-yield, full-length cDNA library has been constructed from canine pancreatic poly(A)+ mRNA. Full-length clones coding for the two major trypsinogen isoenzyme forms have been identified by colony hybridization and verified by in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and an optimal redox potential. Disulfide-bonded translation products were separated and identified by two-dimensional isoelectric focusing-sodium dodecyl sulfate-gel electrophoresis. Nucleotide sequence analysis allowed us to deduce the amino acid sequences for the anionic and cationic forms of canine trypsinogen, which contain 232 and 231 residues, respectively (77% amino acid identity), and the 15-residue amino terminal signal sequences (53% amino acid identity) associated with the two presecretory forms. Measurements of relative and absolute mRNA levels, when related to relative protein synthesis values, indicated that the translational efficiency of anionic trypsinogen mRNA exceeded that of cationic trypsinogen mRNA by 1.5- to 2.9-fold under basal conditions. Analysis of the 5' noncoding regions of trypsinogen mRNAs revealed a striking conservation of sequence (10 of 12 bases) between dog and rat anionic trypsinogen forms. This contrasted markedly with the divergence of the 5' noncoding regions observed between dog anionic and cationic trypsinogen mRNAs.

Amino Acid Sequence↗

PubMed1983

Expression of the native alpha and beta interferon genes in human cells.

We have investigated the expression of human interferon (IFN)-related transcripts in Sendai virus-induced lymphoblastoid (Namalwa) cells and in poly(I) X poly(C)-induced fibroblast (FS-4) cells by electrophoresis of RNA through agarose-CH3HgOH gels followed by (a) blot hybridization of the RNA using IFN-alpha 1 and IFN-beta 1 cDNA probes, and (b) translation of the eluted RNA into biologically active IFN using the Xenopus laevis oocyte assay. In Namalwa cells induced with or without prior treatment with bromodeoxyuridine we can detect IFN-alpha 1 hybridizable, translationally active, cytoplasmic RNA of lengths 0.3-0.6, 2-3, 3.5-5, and 7-8 kb in addition to the expected 0.8-1.4-kb RNA and IFN-beta 1-hybridizable, translationally active, cytoplasmic RNA of lengths 0.9 kb and approximately 4 kb. However the translationally active 1.8-kb IFN-alpha L mRNA(s) in Namalwa cells (P. B. Sehgal, May, LaForge, and Inouye, Proc. Nat. Acad. Sci. USA 79, 6932-6936, 1982) appears to have less than 60-70% nucleotide sequence homology with IFN-alpha 1 in the coding region because it does not cross-hybridize IFN-alpha 1 cDNA even under very relaxed hybridization conditions. In induced FS-4 cells we can detect IFN-beta 1-hybridizable and translationally active cellular RNA of lengths 0.3-0.4, 0.65, 0.9, 1.8, 2-3, 3.5-5, and 7-8 kb. No hybridization is detected in RNA of length 1.3 kb ("IFN-beta 2 mRNA"). Direct comparisons between the translation and hybridization data using dot-blot hybridization procedures confirm that the apparently subcistronic 0.35-kb IFN-beta 1 hybridizable-RNA species detected in FS-4 cells is translationally active.

Bromodeoxyuridine↗

PubMed1983

Isolation of novel human genomic DNA clones related to human interferon-beta 1 cDNA.

Southern blot-hybridization analyses of human DNA (from Namalwa lymphoblastoid cells) digested with the restriction endonuclease EcoRI were carried out under optimal conditions with two human fibroblast interferon (IFN-beta 1) cDNA probes, pD19 and pD24, which contain IFN-beta 1 inserts 0.8 and 0.7 kilobase (kb) long, respectively. The analyses revealed the presence of several hybridizable DNA fragments, including two of lengths 6.8 and 5.5 kb, in addition to the classical IFN-beta 1 genomic DNA fragment of length approximately equal to 2.0 kb. We have screened a human DNA library in lambda bacteriophage Charon 4A by using a 32P-labeled IFN-beta 1 insert cDNA (pD24) and thereby isolated six strongly positive human genomic DNA clones. One of these (lambda B37) represents the classical human IFN-beta 1 gene; another (lambda B37) contains a 6.8-kb EcoRI DNA fragment(s) which cross-hybridizes with the IFN-beta 1 cDNA insert probes pD19 and pD24; and the remaining four (which are identical to each other and are exemplified by lambda B4) contain two EcoRI DNA fragments approximately 5.5 and 9 kb long which also cross-hybridize the IFN-beta 1 cDNA probes. A mRNA 0.9 kb long derived from the classical IFN-beta1 gene is expressed in poly(I) . poly(C)-induced human diploid fibroblasts (FS-4 strain). Induced FS-4 cells also contain polyadenylylated RNA 1.8, 3, 5, and approximately equal to 8 kb long derived from the lambda B3 gene, all of which appear to code for biologically active human IFN-beta as tested by using the Xenopus laevis oocyte translation assay. These data strongly indicate that lambda B3 represents a novel functional IFN-beta gene. A 12-kb polyadenylylated RNA, derived from lambda B4, is expressed constitutively at a low level in FS-4 cells, but the amount of this RNA increases 5-7 hr after exposure of the cells to poly(I) . poly(C).

Base Sequence↗

PubMed1983

Identification of cDNA clones encoding secretory isoenzyme forms: sequence determination of canine pancreatic prechymotrypsinogen 2 mRNA.

A cDNA library has been constructed from canine poly(A)+ mRNA. Clones containing cDNA inserts coding for prechymotrypsinogen 2 (isoelectric point = 7.1; Mr = 27,500), one of three canine pancreatic isoenzyme forms, were selected by colony hybridization using a cDNA probe synthesized from immunoselected prechymotrypsinogen 2 mRNA. To verify that cDNA clones code for prechymotrypsinogen 2 forms that translocate across rough endoplasmic reticulum membranes and fold into stable and identifiable secretory proteins, we conducted in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and optimal concentrations of glutathione and analyzed nascent translation products in their nonreduced state by two-dimensional isoelectric focusing/NaDodSO4 gel electrophoresis and fluorography. A near full-length chymotrypsinogen 2 cDNA and its primed extension were used to determine the nucleotide sequence for the entire coding region of prechymotrypsinogen 2 mRNA and 87 residues, including a poly(A) addition signal, in the 3' nontranslated region. The deduced amino acid sequence shows a 263-residue presecretory protein containing an 18-residue amino-terminal transport peptide (Met-Ala-Phe-Leu-Trp-Leu-Leu-Ser-Cys-Phe-Ala-Leu-Leu-Gly-Thr-Ala-Phe-Gly ), which we have previously shown to mediate the translocation of chymotrypsinogen 2 across the rough endoplasmic reticulum membrane. Following the transport peptide is a 245-residue proenzyme, which shows 82% and 80% sequence identity with bovine chymotrypsinogens A and B, respectively. Conserved among the three zymogens are 10 Cys residues that form five disulfide bonds in bovine chymotrypsinogens A and B and the residues that are required for zymogen activation, substrate binding, and catalytic activity.

Amino Acid Sequence↗

PubMed1982

Unusually long mRNA species coding for human alpha and beta interferons.

Each of the several intronless interferon (IFN) alpha S genes is embedded in 4- to 5-kilobase (kb) DNA homology units which are arranged more or less in tandem in the human genome. In addition to the expected 0.8- to 1.4-kb-long IFN-alpha S mRNA species, we have detected polyadenylylated RNA molecules approximately 2-2.7, 3.5-4.5, and 7-8 kb long derived from IFN-alpha S genes in the cytoplasm of Sendai virus-induced bromodeoxyuridine-treated human lymphoblastoid (Namalwa) cells. These transcripts were detected by electrophoresis of cytoplasmic polyadenylylated RNA through agarose-CH3HgOH gels followed by blothybridization using an IFN-alpha 1 (a prototype alpha S) cDNA probe and also by translation of the eluted RNA into biologically active IFN in the Xenopus oocyte assay. At least some of these long translationally active RNA molecules appear to represent functional mRNA species because they can be detected in polysomes. In addition, the oocyte translation assay has revealed the presence of unusually long (2-8 kb) IFN-beta mRNA species both in Sendai virus-induced Namalwa cells and in poly(I) . poly(C)-induced diploid human fibroblasts (FS-4). The detection of very long IFN-alpha 1-related mRNA species suggests that some of the DNA sequence features observed in the flanking homology units may affect the transcription of the IFN-alpha S genes. Our hybridization analyses also indicate that the 1.8-kb human IFN-alpha L mRNA(s) shows no detectable cross-hybridization with an IFN-alpha 1 cDNA probe. Thus, this mRNA is derived from a gene(s) that is distinct from the IFN-alpha S set.

Cell Line↗