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At least 721 records · Page 40Linked to original sources
PubMed1997

Molecular cloning of cDNA species for rat and mouse liver alpha-methylacyl-CoA racemases.

cDNA species coding for alpha-methylacyl-CoA racemase were cloned from rat and mouse liver cDNA libraries and characterized. The rat liver lambdagt11 cDNA expression library was screened with anti-racemase IgG [Schmitz, Albers, Fingerhut and Conzelmann (1995) Eur. J. Biochem.231, 815-822]. Several full-length clones were obtained that contained an open reading frame of 1083 bp, coding for a protein of 361 amino acid residues with a predicted molecular mass of 39679 Da. The sequences of three peptides that were isolated by HPLC from a tryptic digest of purified rat liver racemase fully matched the cDNA-derived amino acid sequence. The cDNA coding for mouse racemase was cloned from a mouse liver lambdaZAP cDNA expression library and sequenced. The coding region of 1080 bp codes for a 360-residue protein (molecular mass 39558 Da) that shares 89.7% similarity with the rat protein. Expression of the rat racemase as are combinant protein in Escherichia coli with the pTrcHisB-expression vector yielded enzymically active protein. The amino acid sequences of alpha-methylacyl-CoA racemases do not resemble any known sequence of beta-oxidation or auxiliary enzymes, supporting the view of a highly diverse evolutionary origin of enzymes acting on fatty acyl-CoA S-esters.

Amino Acid Sequence↗
PubMed1998

Developing microsatellite markers from cDNA: a tool for adding expressed sequence tags to the genetic linkage map of the chicken.

A chicken embryonic cDNA library was screened with a (TG)13 probe in order to develop polymorphic microsatellite markers. The redundancy of the embryonic cDNA library with a chicken brain cDNA library, which was used for microsatellite development in a previous study, was extremely high. Of the 300 (TG)13 positive clones, only 80 were unique for the embryonic cDNA library. Still, nine expressed sequences derived from the embryonic cDNA library were mapped in the Wageningen (WAU) resource population. In addition seven microsatellite markers from the chicken brain cDNA library, which were monomorphic or unlinked in the two international reference families in the previous study, were also mapped in the WAU population. Three of the 16 mapped chicken expressed sequence tags (ESTs) showed relatively high percentages of sequence similarity to sequences found in other species. As two of these genes, RAB6 and ZFX/ZFY, have been mapped in humans, they contribute to the comparative map of the chicken.

Animals↗
PubMed1999

Regional insertional mutagenesis of genes on Arabidopsis thaliana chromosome V using the Ac/Ds transposon in combination with a cDNA scanning method.

For regional insertional mutagenesis of Arabidopsis thaliana genes, we combined a cDNA scanning method (Hayashida et al. Gene 1995; 165:155-161) and an Ac/Ds transposon designed for local mutagenesis, and evaluated this approach with two overlapping yeast artificial chromosome (YAC) clones, CIC7E11 and CIC8B11, on A. thaliana chromosome 5. We applied a previously developed novel cDNA selection method using DNA latex particles (cDNA scanning method) to the two YAC clones and constructed two sub-libraries in which cDNAs for genes on each YAC DNA were concentrated. From each sub-library we isolated cDNAs for genes on each YAC DNA, partially sequenced them, and produced expressed sequence tags (ESTs). In total, 113 non-redundant groups of cDNAs were obtained. Forty-four per cent of these EST clones were novel, and 34% had significant homology to functional proteins from various organisms. In parallel, we transposed Ds from a donor Ds-GUS-T-DNA line, Ds4391-20, already mapped to the CIC7E11/8B11 region. We obtained Ds-transposed lines and recovered their Ds-flanking genomic DNAs by thermal asymmetric interlaced (TAIL) polymerase chain reaction (PCR). Dot-blot analysis indicated that 20% of the lines contained transposed Ds in the CIC7E11/8B11 region, suggesting that this Ac/Ds transposon system is effective for regional insertional mutagenesis. To isolate Ds insertion mutants in the genes identified from the CIC7E11/8B11 region, we carried out PCR screening from 800 Ds-containing lines using Ds-specific and gene-specific primers that were designed from the 113 cDNA sequences identified by the cDNA scanning method. We found that 49 lines contain Ds insertion mutations, and that five lines contain Ds mutations in genes that are mapped to the sequenced CIC7E11/8B11 genomic DNA region. These results indicate that combining the cDNA scanning method and the Ac/Ds transposon gives a powerful tool for regional insertional mutagenesis not only in Arabidopsis but also in other plants or crops whose genomes are not sequenced.

Arabidopsis↗
PubMed2001

Mapping genes on an integrated sorghum genetic and physical map using cDNA selection technology.

Sorghum is an important target of plant genomics. This cereal has unusual tolerance to adverse environments, a small genome (750 Mbp) relative to most other grasses, a diverse germplasm, and utility for comparative genomics with rice, maize and other grasses. In this study, a modified cDNA selection protocol was developed to aid the discovery and mapping of genes across an integrated genetic and physical map of the sorghum genome. BAC DNA from the sorghum genome map was isolated and covalently bound in arrayed tubes for efficient liquid handling. Amplifiable cDNA sequence tags were isolated by hybridization to individual sorghum BACs, cloned and sequenced. Analysis of a fully sequenced sorghum BAC indicated that about 80% of known or predicted genes were detected in the sequence tags, including multiple tags from different regions of individual genes. Data from cDNA selection using the fully sequenced BAC indicate that the occurrence of mislocated cDNA tags is very low. Analysis of 35 BACs (5.25 Mb) from sorghum linkage group B revealed (and therefore mapped) two sorghum genes and 58 sorghum ESTs. Additionally, 31 cDNA tags that had significant homologies to genes from other species were also isolated. The modified cDNA selection procedure described here will be useful for genome-wide gene discovery and EST mapping in sorghum, and for comparative genomics of sorghum, rice, maize and other grasses.

Base Sequence↗
PubMed1999

Cloning, sequencing and functional expression of a cDNA encoding porcine pancreatic preprocarboxypeptidase A1.

A full-length cDNA clone coding for porcine pancreatic preprocarboxypeptidase A1 (prePCPA1) was isolated from a cDNA library. The open reading frame (ORF) of the nucleotide sequence was 1260 nt in length and encoded a protein of 419 amino acids (aa). The cDNA included a short signal peptide of 16 aa and a 94 aa-long activation segment. The calculated molecular mass of the mature proenzyme was 45561 Da, in accordance with that of the purified porcine pancreatic PCPA1. The deduced aa sequence of the corresponding enzyme differed from that predicted by the three-dimensional structure by 40 aa, and showed 85% identity and 55% identity to that of procarboxypeptidases A1 and A2, respectively. Moreover the sequence was identical to that of several independent cDNA clones, suggesting that it is the major transcribed gene. No evidence for a second variant was observed in the cDNA library and PCPA2 is apparently absent from the porcine pancreas. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast triose phosphate isomerase promoter. The signal peptide of the PCPA protein efficiently directed its secretion into the culture medium (1.5 mg.L-1) as a protein of the predicted size. The recombinant proenzyme was analyzed by immunological and enzymological methods. Its activation behavior was comparable with that of the native form and led to a 35-kDa active enzyme.

Animals↗
PubMed2000

cDNA cloning, genomic cloning, and tissue-specific regulation of mouse cerebroside sulfotransferase.

We have isolated a mouse cDNA clone encoding 3'-phosphoadenylylsulfate-galactosylceramide 3'-sulfotransferase (cerebroside sulfotransferase; CST; EC 2.8.2.11) from a kidney cDNA library, using a human CST cDNA clone [Honke, K., Tsuda, M., Hirahara, Y., Ishii, A., Makita, A. & Wada, Y. (1997) J. Biol. Chem. 272, 4864-4868] as a probe. A recombinant protein of the cloned cDNA showed CST activity. The deduced protein is composed of the same 423 amino acids as human CST and its sequence exhibits 84% identity with that of the human counterpart. Northern-blot analysis and subquantitative reverse transcription-PCR (RT-PCR) analysis showed that the CST gene is preferentially transcribed in stomach, small intestine, brain, kidney, lung, and testis, in that order. To examine differences in transcripts in various tissues, we isolated CST cDNA clones from stomach, small intestine, brain, kidney, and testis by 5'-RACE analysis. We found seven different nucleotide sequences in the 5'-UTR, while the DNA sequences of all the isolated cDNA clones were identical in the coding region. In addition, we isolated CST genomic DNA clones from a mouse genomic library. The clones covered all the 5'-UTR sequences and coding exons including 3'-UTR. RT-PCR analyses of CST mRNAs from various tissues confirmed that CST transcripts are tissue-specifically spliced by alternative use of multiple exons 1. These observations suggest that the tissue-specific expression of the CST gene is explained by alternative usage of multiple 5'-UTR exons flanked with tissue-specific promoters.

Amino Acid Sequence↗
PubMed1997

Molecular cloning of a peptidase against N-acetylaspartylglutamate from a rat hippocampal cDNA library.

N-Acetylaspartylglutamate (NAAG) is the most prevalent peptide neurotransmitter in the mammalian nervous system. NAAG selectively activates the type 3 metabotropic glutamate receptor. It is inactivated by peptidase activity on the extracellular face of the plasma membrane of neurons and glia. The human gene that codes for prostate-specific membrane antigen (PSM) has been shown to produce peptidase activity against NAAG. We cloned the human PSM cDNA and used it to probe a rat hippocampal cDNA library. We identified a cDNA containing a complete coding region that possesses 83% homology with the PSM gene. The predicted 752-amino acid sequence has 85% identity and 91% similarity to the PSM sequence. CHO cells transfected with this cDNA expressed NAAG peptidase activity at a level similar to that obtained from rat brain membranes. The peptidase activity was inhibited by beta-NAAG, quisqualate, and pteroylglutamate but not aspartylglutamate or pteroic acid. In situ hybridization data demonstrated the widespread distribution of the peptidase mRNA in the brain, consistent with the distribution of peptidase activity. The highest levels of hybridization were detected in the hippocampus, dentate gyrus, piriform cortex, choroid plexus of the ventricles, pineal gland, anterior pituitary, and supraoptic nucleus. Three transcripts (estimated at 5, 3.4, and 2.9 kb) were identified in northern blots of rat brain, while in rat kidney the third transcript appeared slightly smaller than 2.9 kb. With use of reverse transcriptase PCR with primers for the 5' end, the central region, and the 3' end of the hippocampal cDNA, the expected amplification products were obtained from rat brain RNA. Spinal cord yielded an amplification product only with primers for the 5' end of the hippocampal cDNA.

Amino Acid Sequence↗
PubMedDate not supplied

Cloning and sequencing of the complete cDNA encoding the human insulin receptor related receptor.

The insulin receptor related receptor (IRR) is a heterotetrameric transmembrane receptor with intrinsic tyrosine kinase activity. The IRR shares large homology with the insulin and the insulin-like growth factor-1 (IGF-I) receptor with regard to amino acid sequence and protein structure. So far, only a partial human sequence containing the complete 3' end has been reported, although the full-length human IRR cDNA had been used for transfection studies and functional analysis of the receptor. We have isolated a full-length human IRR cDNA and report on the 5' translated and untranslated region of the human IRR gene. The full length IRR sequence contains 4150 bases and shares a high degree of homology with the guinea pig IRR cDNA sequence and rat IRR sequences that had been reported earlier on by others. Sequencing of the IRR cDNA revealed that the human IRR cDNA contains 341 bases corresponding to the IRR 5' end in addition to the bases that had been reported on before. Also, this sequence contains the start codon of translation. The full length cDNA for the human IRR can now be used for functional expression studies and to elucidate the nature of the ligand for this receptor type.

Cloning, Molecular↗
PubMed2001

cDNA cloning and immunologic characterization of Pen o 18, the vacuolar serine protease major allergen of Penicillium oxalicum.

Penicillium species are prevalent indoor airborne fungi that have been identified as causative agents of human extrinsic bronchial asthma. In the preparation of standardized diagnostic reagents, it is imperative to define the allergens of these ubiquitous fungi. Results from our previous study on P. oxalicum suggest that the 34-kd major immunoglobulin E-reacting component of this prevalent Penicillium species is probably a vacuolar serine protease. The purpose of the present study was to define this major P. oxalicum allergen (Pen o 18) through cDNA cloning and immunologic characterization. The cDNA of Pen o 18 was isolated through a combination of reverse transcriptase-polymerase chain reaction and 5'- and 3'-rapid amplification cDNA ends reactions. The primers used in these reactions were constructed according to the internal amino acid sequences of Pen o 18 and the conserved amino acid sequences of fungal serine proteases. Our results showed that a 1897-bp cDNA with an open reading frame of 503 residues was isolated for the proenzyme of Pen o 18. The encoded protein has a 16-residue signal peptide and a 119-residue prosequence. On maturation, the protein has an N-terminal glutamate that is the 136th residue encoded by the cDNA. Apparently the precursor also undergoes C-terminal processing with the cleavage of about 47 amino acids. The cDNA for Pen c 18 (the vacuolar serine protease allergen from P. citrinum ) was also isolated for comparison. Contrary to a previous report, the C-terminal region of Pen c 18 is similar to that of Pen o 18. Recombinant proteins (rPen o 18 and rPen c 18) with the putative mature N-termini and a his-tag were obtained by expressing the corresponding cDNAs in Escherichia coli. Serum samples from 7 asthmatic patients with immunoglobulin E reactivity to the 34-kd component of P. oxalicum also react to his-tagged recombinant Pen o 18. The presence of immunoglobulin E cross-reactivity between rPen o 18 and rPen c 18 was detected by immunoblot inhibition. Two monoclonal antibodies (PCM39 and FUM20) against fungal serine proteases react with rPen o 18, rPen c 18, and the 35/34-kd components in the corresponding crude fungal extracts. These components also react with immunoglobulin E antibodies in serum samples from asthmatic patients. In conclusion, results obtained confirm that the 34-kd major allergen of P. oxalicum is a vacuolar serine protease. The cDNAs of Pen o 18 and Pen c 18 encode precursor molecules that appear to undergo both N-terminal and C-terminal processing. Constructs beginning with mature N-terminal can be expressed in E. coli to produce recombinant polypeptides that are reactive to monoclonal antibodies or immunoglobulin E antibodies in serum samples from asthmatic patients. Results obtained may provide useful information and materials for preparation of standardized diagnostic reagents in clinical mold allergy.

Adult↗
PubMed1982

Molecular cloning and nucleotide sequence of full-length of cDNA coding for porcine gastrin.

We have cloned in EScherichia coli a cDNA copy of mRNA coding for the porcine antral mucosal hormone preprogastrin. Full-length double-stranded cDNA was synthesized and inserted into the Pst I endonuclease site in plasmid pBR322 by using a homopolymeric extension technique. A partial cDNA clone was used as a probe to identify a complete cDNA clone in a cDNA library by colony hybridization. Four positive clones were isolated, one of which corresponded to porcine preprogastrin mRNA. The nucleotide sequence of the cDNA insert (602 nucleotides) revealed 312 nucleotides in the entire mRNA coding region, 61 nucleotides in the 5' untranslated region, 86 nucleotides in the 3'untranslated region, and a poly(A) tail of 86 nucleotides. Gastrin is located near the carboxyl end of preprogastrin and is flanked at both its amino and carboxyl ends by a pair of basic amino acid residues. The presence of glycine and a pair of basic amino acid residues adjacent in the carboxyl-terminal phenylalanine of gastrin indicates that the glycine and a pair of basic amino acid residues may be required for the enzymatic amidation of phenylalanine to phenylalanine amide.

Animals↗
PubMed1983

Isolation of cDNA clones specific for collagen IV and laminin from mouse teratocarcinoma cells.

The synthesis of the proteins laminin and collagen IV is stimulated approximately equal to 20-fold in F9 mouse teratocarcinoma stem cells after treatment of the cells with retinoic acid and N6, O2'-dibutyryl-cAMP (Bt2cAMP). A cDNA library from F9 cells treated with retinoic acid, Bt2cAMP, and theophylline (F9-R + DBC cells) was constructed to isolate cDNA coding for collagen IV or laminin. The recombinant plasmids were screened by differential colony hybridization to cDNA synthesized from poly(A)+ RNA isolated from F9 stem and F9-R + DBC cells. Differentially hybridizing plasmids were then used as probes to hybridize to RNA transfer blots to determine the size of their specific mRNA. Only plasmids containing cDNA sequences specific for high molecular weight mRNA were further analyzed. Studies by hybridization-selection, in vitro translation, and immunoprecipitation showed that a plasmid clone, pc15, contains cDNA homologous to collagen IV (alpha 2) mRNA, and another plasmid clone, pc156, contains cDNA homologous to laminin B mRNA. By RNA blot analyses, the size of mRNA coding for collagen IV (alpha 2) is 7.6 kilobases; the size of mRNA for laminin is 6.8 kilobases. Using the technique of RNA blot hybridization, we studied the time course of the increase in mRNA coding for collagen IV (alpha 2) and laminin B in F9 cells after retinoic acid and Bt2cAMP treatment. Both collagen IV (alpha 2) and laminin B mRNAs are present in F9 stem cells. Collagen IV (alpha 2) mRNA and laminin B mRNA levels increase slightly at approximately 12 hr after retinoic acid and Bt2cAMP addition, with a dramatic increase between 12 and 24 hr after drug treatment.

Animals↗
PubMed1984

Isolation of cloned cDNA encoding mammalian ornithine decarboxylase.

During the growth of mammalian cells the level of ornithine decarboxylase ( OrnDCase ; L-ornithine carboxy-lyase, EC 4.1.1.17), the first enzyme in polyamine biosynthesis, undergoes rapid changes. As an initial step in the study of possible genetic mechanisms involved in these changes, we have isolated cDNA clones encoding OrnDCase . To obtain RNA enriched for OrnDCase messenger, mouse myeloma cells that overproduce OrnDCase were selected in the presence of the OrnDCase inhibitor, difluoromethylornithine. A pBR322 cDNA library was prepared from poly(A)+ RNA isolated from difluoromethylornithine-resistant cells, and the library was probed with [32P]cDNA representing mRNA sequences from resistant or parental (sensitive) cells. All clones hybridizing preferentially to the resistant cell probe shared nucleotide sequences. A representative clone containing 1.1 kilobases of cDNA was shown to encode OrnDCase sequences by in vitro translation of hybrid-selected mRNA followed by precipitation of the translation products with anti- OrnDCase antiserum. Using this cDNA clone as a probe, we found that mouse DNA yielded several restriction fragments that react with the OrnDCase cDNA. In the difluoromethylornithine-resistant myeloma cells, one of these DNA segments is amplified and the level of OrnDCase mRNA is greatly increased compared with that in parental plasmacytoma cells. The level of OrnDCase mRNA is also increased in cultured 3T3 cells stimulated with serum and in mouse kidneys after administration of androgen, indicating that OrnDCase gene transcription and/or mRNA stability are regulated during cell growth.

Animals↗
PubMed1984

Molecular cloning and characterization of a cDNA for the beta subunit of human alcohol dehydrogenase.

Human alcohol dehydrogenase (ADH) is encoded by at least five genes that fall into three classes. The class I ADH genes encode the three closely related alpha, beta, and gamma polypeptides. Molecular genetic analysis of class I ADH genes has been initiated by isolating a cDNA clone from a human adult liver cDNA library. A synthetic oligonucleotide mixture encoding a portion of the beta subunit of ADH was used as an in situ hybridization probe for the cDNA library. One positively hybridizing clone, pADH12, which contained an 1100-base-pair cDNA insert, was subjected to DNA sequence analysis. The sequence indicated that the cDNA encoded information for the carboxyl-terminal 91 amino acids of a class I ADH and a 3' untranslated region of 593 nucleotides. Comparisons with the carboxyl terminus of the human ADH beta subunit indicated that the cDNA encoded the beta polypeptide. This probe may facilitate genetic studies of various human alcohol-related syndromes, as well as enable basic molecular studies on human ADH gene expression.

Adult↗
PubMed1984

Molecular cloning of a cDNA sequence complementary to porphobilinogen deaminase mRNA from rat.

A cDNA clone containing sequences complementary to the mRNA coding for anemic rat spleen porphobilinogen deaminase (EC 4.3.1.8) has been isolated. A cDNA library was prepared from partially purified mRNA (1% purity). This library was then screened by colony hybridization, using a cDNA probe derived from porphobilinogen deaminase mRNA further enriched (10-20% purity) by gel electrophoresis in the presence of methylmercury hydroxide. Colonies hybridizing with the probe were analyzed by hybrid-selected translation using anemic rat spleen mRNA. Four recombinant plasmids containing porphobilinogen deaminase cDNA sequences were identified by specific immunoprecipitation of the translational product from hybrid-selected mRNA. Porphobilinogen deaminase mRNA was shown to contain 1800 bases by blot hybridization analysis. The cloned cDNA sequence consists of 1500 bases. Hybridization analysis of poly(A)+ RNA from uninduced and induced mouse erythroleukemic cells indicated that induction to erythroid differentiation by dimethyl sulfoxide results in a 10-fold increase of porphobilinogen deaminase mRNA. The rat cDNA clones hybridize to the corresponding sequences encoding human porphobilinogen deaminase. This property will be useful for isolation of human gene(s) and further characterization of the molecular lesion(s) responsible for acute intermittent porphyria.

Ammonia-Lyases↗
PubMed1985

Amino acid sequence of the alpha subunit of transducin deduced from the cDNA sequence.

Transducin, a GTP-binding protein involved in phototransduction in the vertebrate retina, belongs to a family of homologous coupling proteins that also includes Gs and Gi, the regulatory proteins of adenylate cyclase. Here we report the cDNA sequence and deduced amino acid sequence of transducin's alpha subunit (T alpha). The cDNA was isolated, by screening with an antibody probe, from a bovine retinal cDNA library in the expression vector lambda gt11. The 2.2-kilobase cDNA insert hybridized to a single 2.6-kilobase poly(A)+ RNA species present in extracts of bovine retina but not of bovine heart, liver, or brain. The nucleotide sequence of the cDNA revealed an open reading frame long enough to encode the entire 39-kDa T alpha polypeptide. The polypeptide sequence deduced from the cDNA would be composed of 350 amino acids and have a molecular weight of 39,971. Portions of the sequence matched reported amino acid sequences of T alpha tryptic fragments, including sites specifically ADP-ribosylated by cholera and pertussis toxins. The predicted sequence also includes four segments, ranging from 11 to 19 residues in length, that exhibit significant homology to sequences of GTP-binding proteins, including the ras proteins of man and yeast and the elongation factors of ribosomal protein synthesis in bacteria, EF-G and EF-Tu. In combination with previous functional studies of tryptic fragments of T alpha, the deduced amino acid sequence makes it possible to predict which portions of the polypeptide interact with other molecules involved in retinal phototransduction.

Amino Acid Sequence↗
PubMed1985

Cloning and expression of cDNA for rat heme oxygenase.

Two cDNA clones for rat heme oxygenase have been isolated from a rat spleen cDNA library in lambda gt11 by immunological screening using a specific polyclonal antibody. One of these clones has an insert of 1530 nucleotides that contains the entire protein-coding region. To confirm that the isolated cDNA encodes heme oxygenase, we transfected monkey kidney cells (COS-7) with the cDNA carried in a simian virus 40 vector. The heme oxygenase was highly expressed in endoplasmic reticulum of transfected cells. The nucleotide sequence of the cloned cDNA was determined and the primary structure of heme oxygenase was deduced. Heme oxygenase is composed of 289 amino acids and has one hydrophobic segment at its carboxyl terminus, which is probably important for the insertion of heme oxygenase into endoplasmic reticulum. The cloned cDNA was used to analyze the induction of heme oxygenase in rat liver by treatment with CoCl2 or with hemin. RNA blot analysis showed that both CoCl2 and hemin increased the amount of hybridizable mRNA, suggesting that these substances may act at the transcriptional level to increase the amount of heme oxygenase.

Amino Acid Sequence↗
PubMed1985

Murine plasma cell membrane antigen PC-1: molecular cloning of cDNA and analysis of expression.

PC-1 is a membrane glycoprotein expressed selectively on murine antibody-secreting plasma cells. Previously, we have obtained partial amino acid sequence data for this protein. Here, we describe the use of these data in the isolation of PC-1 cDNA clones. To avoid the "3'-bias" of conventional cDNA libraries we constructed a cDNA library in lambda gt10 by priming the first strand of cDNA synthesis with random hexadeoxynucleotides. The library was screened with oligonucleotide probes, 17 nucleotides in length, the design of which was based on the amino acid sequence data. Two cDNA clones of 1.0 and 0.9 kilobase pairs (lambda RR3 and lambda RR20, respectively) were isolated. Both contained a sequence encoding the 15-residue tryptic peptide that was used to derive the sequences of the oligonucleotide probes. lambda RR3 cDNA hybridized to an approximately equal to 3.5-kilobase mRNA that was present in plasmacytomas, spleen, and liver but not in other cell types screened. We were unable to detect PC-1 mRNA or protein in mouse brain. In the spleens of mice chronically infected with Mesocestoides corti, PC-1 mRNA was present at 2.5-fold higher levels than found in normal mouse spleens, whereas immunoglobulin mRNA levels were 15-fold higher. Southern blot analyses revealed the presence of only one gene copy per haploid mouse genome. Restriction fragment length polymorphisms were detected in genomic DNA from mice bearing different PC-1 alleles. A related gene is present in rat genomic DNA.

Animals↗
PubMed1985

Isolation of a cDNA clone encoding pancreatic polypeptide.

We have isolated a cDNA clone encoding pancreatic polypeptide from a cDNA library constructed with RNA from an endocrine neoplasm of the human pancreas. The cDNA was inserted into plasmid pBR322 and the plasmid was cloned in Escherichia coli. Oligonucleotides (sequence in text) specific for the amino acid sequence (sequence in text) of pancreatic polypeptide were used as hybridization probes. The pancreatic polypeptide cDNA isolated was 465 base pairs long and encoded a peptide of 95 amino acids in the coding region. The 36-amino acid sequence of pancreatic polypeptide was flanked by a 29-amino acid putative leader sequence at the amino terminus and a connecting tripeptide (Gly-Lys-Arg) followed by a 27-amino acid peptide at the carboxyl terminus. The first 20 of the amino acids in the carboxyl-terminal heptacosapeptide were identical to the structure of human pancreatic icosapeptide with the single exception of an isoleucine substitution for valine in the 18th position. This alteration results from an A----G substitution in the nucleotide sequence of the cDNA and may represent a genetic variation or a point mutation in the pancreatic polypeptide cDNA.

Amino Acid Sequence↗