Characterization of a complementary deoxyribonucleic acid for the coagulogen of Limulus polyphemus

An 869-nucleotide-long cDNA clone for the coagulogen from Limulus amebocyte has been isolated and its nucleotide sequence has been determined. The deduced amino-acid sequence revealed a signal peptide, 20 amino acids long, and a mature protein of 175 amino acids. The amino-acid sequence of the coagulogen was compared to all known proteins by two computer programs. Using these programs, Limulus coagulogen showed 70% homology with the coagulogen of Tachypleus tridentatus (Japanese horseshoe crab). Further computer analysis showed no statistically significant homology to support an evolutionary origin of the horseshoe crab coagulogen common to other protein families. These results place horseshoe crab coagulogen in a new superfamily unrelated to any other proteins investigated. RNA blot analysis of Limulus RNA indicated that the coagulogen mRNA was about 900 bases long and represented an abundant species in the amebocyte while detected only in small quantities in the hepatopancreas. Besides mature RNA, high-molecular-weight forms of coagulogen RNA were also observed. Southern blot analysis of Limulus DNA digested with restriction endonucleases suggested that the Limulus coagulogen gene contains at least three introns, or belongs to a multigene family.     

Characterization of complementary deoxyribonucleic acid for precursor of porcine motilin

Cloned cDNAs encoding the precursor protein for motilin and a novel peptide, motilin-associated peptide, were isolated from a library derived from porcine intestinal mucosa mRNA. Nucleotide sequence analysis predicts a precursor protein of 119 amino acids including a signal peptide in direct linkage with the 22 amino acid sequence for motilin, and a 70 amino acid peptide of unknown function. The putative bioactive moieties are separated by Lys-Lys, dibasic residues that serve as substrates for cleavage by proteolytic maturation enzymes in many polyprotein precursors. While there is an abundant literature detailing a spectrum of tissues and cell types which express motilin like immunoreactivity, analysis of mRNA derived from many of these tissues suggests that the mRNA for the mucosal motilin precursor is only transcribed in this tissue. The nature of the immunoreactive material in the central nervous system and other peripheral tissues remains to be determined.     

Multiplicity of deoxyribonucleic acid sequences with homology to a cloned complementary deoxyribonucleic acid coding for rat phenobarbital-inducible cytochrome P-450

We previously identified cDNA clones for rat cytochrome P-450 of the phenobarbital-inducible type by sequence analysis [Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., & Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2793-2797]. With these cloned cDNAs as probe, the multiplicity of phenobarbital-inducible cytochrome P-450 gene in rat genome was investigated by three approaches. The first approach was the Cot analysis of the total rat liver DNA under conditions of DNA excess. With internal and external markers used as gene-number standards, the reassociation kinetics were studied, which suggested the presence of approximately six genes or gene-like sequences hybridizable to phenobarbital-inducible cytochrome P-450 cDNA per rat haploid genome. The second was the isolation of the cytochrome P-450 genes from a rat genomic library. From a screening of about 1 X 10(6) plaques, nine clones with an approximately 15-kb insert were isolated. Restriction maps and Southern blot analysis of the cloned DNAs showed that six out of nine isolated clones contained DNA inserts independent of one another. The third was Southern blot analysis of rat genomic DNA with restriction enzyme EcoRI. Approximately 12 positive bands were demonstrated with the cDNA probe, seven to eight of which showed the same mobilities as the fragments in the isolated six genomic clones, suggesting that some other genes or gene-like DNA sequences remained to be cloned.     

Cloning and nucleotide sequence analysis of complementary deoxyribonucleic acid for bovine preproinsulin

A cDNA library was prepared from poly(A+) RNA isolated from fetal bovine pancreas. Bacterial colonies were screened for sequences homologous to a rat preproinsulin I cDNA probe. Ten positive clones were selected at random and further studied. Northern blot analyses revealed that seven of these clones hybridized to a single RNA species, of approximately 400 nucleotides. Sequence analysis of one of these clones (pbI2885) revealed the entire structural region of bovine preproinsulin mRNA including a 72 nucleotide region encoding a signal peptide enriched in hydrophobic residues. The overall nucleotide homology between bovine and human preproinsulin mRNA was 76% for the preregion, 89% for the A chain, 83% for the B chain, and 68% for the C peptide (including a 15 nucleotide deletion).     

Characterization of complementary deoxyribonucleic acid for human adrenocortical 17 alpha-hydroxylase: a probe for analysis of 17 alpha-hydroxylase deficiency

To provide a basis for investigation of the molecular mechanisms underlying the hormonal regulation of steroid 17 alpha-hydroxylase (P-450 17 alpha) activity in adrenal, ovary, and testis as well as human 17 alpha-hydroxylase deficiency, we have isolated from a human fetal adrenal cDNA library a cDNA sequence complementary to the mRNA that encodes the human P-450 17 alpha enzyme. Of 75,000 colonies from the library that were screened by use of a nick-translated 5'-specific bovine P-450 17 alpha cDNA probe, 10 positive colonies were isolated and the clone with the longest insert (pcD-17 alpha H) was selected for further characterization. pcD-17 alpha H encodes the complete human P-450 17 alpha protein having approximately 78% homology at the nucleotide level and 71% homology at the amino acid level when the sequence of pcD-17 alpha H is compared to the bovine P-450 17 alpha cDNA sequence. By transient expression of the human P-450 17 alpha cDNA clone in COS 1 cells, we have demonstrated that the 17 alpha-hydroxylase and 17,20 lyase activities reside within the same human P-450 17 alpha polypeptide chain. The insert was also used as a probe to investigate, by means of Southern blot analysis, possible alterations in the P-450 17 alpha gene sequence in DNA isolated from skin fibroblasts from three patients with clinically characterized 17 alpha-hydroxylase deficiencies. No changes were detected in the DNA of any of the patients by this analysis.     

Nucleotide sequence of cloned complementary deoxyribonucleic acid for the alpha subunit of bovine pituitary glycoprotein hormones

Recombinant DNA plasmids containing sequences coding for the alpha subunit of the bovine pituitary glycoprotein hormones have been isolated. The nucleotide sequences of three different cDNA clones have been determined. The largest alpha-subunit cDNA clone was found to contain 713 bases including 77 nucleotides from the 5'-untranslated region, 72 nucleotides coding for a precursor segment, 288 nucleotides coding for the mature alpha subunit, and 276 nucleotides from the 3'-untranslated region of the mRNA followed by a poly(A) segment. This cDNA likely represents most of the bovine alpha-subunit mRNA sequence. Nucleotide sequences were obtained from the cDNA inserts of two other alpha-subunit clones, and several differences among the three cDNA sequences have been detected. These differences in nucleotide sequence may represent either individual variation in genomic sequence or cloning artifacts. Comparison of the bovine alpha-subunit cDNA sequence to the sequences of human, rat, and mouse alpha-subunit cDNAs reveals that the bovine sequence has greater than 70% homology with the other cDNAs. The cloned alpha-subunit cDNA should provide a useful probe for further studies of the structure and expression of this interesting gene.     

Cloning of a complementary deoxyribonucleic acid coding for human thyroxine-binding globulin (TBG): existence of two TBG messenger ribonucleic acid species possessing different 3'-untranslated regions

An adult human liver cDNA library constructed in expression vector, bacteriophage lambda gt11, was screened with polyclonal antibody directed against human T4-binding globulin (TBG). TBG cDNA cloned in the present study was 944 nucleotides in length. It contained approximately 70% of the coding region and complete 3'-untranslated region. When the sequence was compared with that of TBG cDNA recently cloned by I. L. Flint, T. J. Bailey, T. A., Gustafson, B. E. Markham, and E. Morkin, the 3'-untranslated region of our cDNA was 231 nucleotides shorter than their cDNA. These results indicated that two TBG mRNAs with different length of 3'-untranslated regions may exist in human liver. Indeed, Northern blot analysis revealed that two TBG mRNAs differing in the length approximately 200 base pairs were present in normal human liver as well as in human hepatoma cell line (HepG2). It was demonstrated that this size difference was due to the length of 3'-untranslated region by hybridization with a probe specific to the longer 3'-end. Together with the sequence data, it was suggested that these two TBG mRNA species may be produced by alternative processing and polyadenylation at two different sites.     

Molecular cloning and sequencing of a rat preprogastrin complementary deoxyribonucleic acid

Gastrin biosynthesis involves a complex series of posttranslational modifications; their elucidation requires a knowledge of the structure of the gastrin precursor. The complete structure of rat preprogastrin was deduced from the nucleotide sequence of a full length cDNA clone isolated from a rat antral cDNA library. Northern blot hybridization analysis of rat antral RNA together with human antral RNA, reveals a single mRNA species of approximately 670 bases. Comparison of this sequence with those of porcine and human gastrin reveals extensive (73%) homology in the gastrin coding region as well as short regions of conserved nucleotides in the noncoding regions. The rat sequence encodes a preprogastrin of 104 amino acids which consists of a signal peptide, a 37 amino acid prosegment; and the gastrin 34 sequence, followed by a glycine (the amide donor), and flanked by pairs of arginine residues. Cleavage at an internal pair of lysine residues yields gastrin 17. Unlike the human and porcine sequences, rat preprogastrin contains a 9 amino acid carboxy-terminal extension peptide (-Ser-Ala-Glu-Glu-Glu-Asp-Gln-Tyr-Asn) which is homologous to the midportion of gastrin 17 including the site of tyrosine sulfation.     

Complete amino acid sequence of bovine plasminogen. Comparison with human plasminogen

The amino acid sequence of the single polypeptide chain of bovine plasminogen (786 residues, Mr 88092) was determined. Cleavage with CNBr yielded 13 fragments of which six originated from cleavage sites different from human plasminogen. Digestion with elastase gave three major fragments: kringles (1 + 2 + 3) and kringle 4, both with intact lysine binding sites, and mini-plasminogen. Subfragmentation was achieved mainly with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine (BNPS-skatole), Staphylococcus aureus V8 protease and trypsin. The sequences of fragments which were determined by automated Edman degradation, were aligned with overlapping sequences, or, in a few instances, by homology with the known sequence of human plasminogen. Sequence comparison with the human protein showed varying degrees of homology in the different functional and structural domains. The overall identity (78%) is practically the same as that found in those regions corresponding to the heavy (79%) and the light chain (80%) of plasmin. The average degree of identity among the kringles is 83%. Outside the kringle structures the extent of identity decreases, to 65% in the N-terminal region and to about 50% in the connecting strands between the kringles except for the strand between kringles 2 and 3, where only one out of 12 residues is exchanged. The results reported show that bovine plasminogen apparently contains the same structural and functional domains as human plasminogen. Bovine plasminogen also contains two carbohydrate moieties. The only partially substituted N-glycosidic site, Asn289, corresponds to partially glycosylated Asn288 in human plasminogen, whereas the O-glycosidic site of the human sequence, Thr345, is shifted to Ser339 in bovine plasminogen.     

Molecular hybridization between rat liver deoxyribonucleic acid and complementary ribonucleic acid

RNA (cRNA) was synthesized in vitro on a template of rat liver DNA and its hybridization with rat liver DNA was studied by using the nitrocellulose-filter method. Sonication of the DNA diminished its apparent capacity to hybridize with RNA by about 50%. This is not due to cross-linkage of DNA molecules, because it could be shown that less than 2% of the sonicated DNA was cross-linked. The effect is due instead to the small size of the sonicated DNA molecules. Below a single-stranded molecular weight of 5×10⁵ the DNA showed a progressive loss of capacity to hybridize with decrease in molecular weight. Evidence is presented suggesting that the apparently diminished capacity of the DNA to hybridize is due to loss of hybridized DNA from the membrane filters. When cRNA at concentrations of up to 25μg/ml is annealed with sonicated total DNA, an apparent hybridization saturation value is found at which about 2.5% of the DNA is hybridized with RNA. Increasing the cRNA concentration tenfold brought about the hybridization of a second component of the DNA approximately equal in amount to the first. The renaturation of rat liver DNA was studied by measuring the fall in the extinction at 260nm and two different components of renaturation were observed within the reiterated fraction of DNA. By hybridizing cRNA with different fractions of rat DNA the two components of the hybridization curve are shown to correspond to the two components of the renaturation curve. The conclusion is drawn that at a cRNA concentration of 250μg/ml most of the reiterated fraction of rat liver DNA is hybridized after annealing for 16h under standard conditions (0.30m-sodium chloride–30mm-sodium citrate at 65°C). Even with such a high cRNA concentration little or no hybridization of the slowly renaturing DNA fraction occurs. It is suggested that the most highly reiterated DNA component is poorly transcribed in vitro.     

Construction and characterization of type II collagen complementary deoxyribonucleic acid clones

The mRNA for type II collagen was purified from embryonic chick sternum or from purified sternal chondrocytes with guanidine thiocyanate as the extractant. Double-stranded cDNAs to procollagen mRNAs from sternum were synthesized and dC-tailed. After annealing with PstI-cleaved, dG-tailed pBR322, this DNA was used to transform Escherichia coli X1776. Transformed colonies were screened by colony hybridization to type I and II collagen cDNAs. Clones that preferentially hybridized to type II cDNA were characterized further. Four such cDNA clones, pCgII-2, 3, 10 and 12, with inserts of 400, 320, 260 and 750 bp, have been identified as type II collagen cDNA clones by several criteria, including their preference for hybridizing with type II rather than type I collagen mRNAs in hybrid-selected translation experiments.     

Molecular cloning and characterization of a progesterone-dependent cat endometrial secretory protein complementary deoxyribonucleic acid

In the cat, a group of low molecular weight secretory proteins have previously been shown to appear in the endometrium after progesterone (P) administration to an estrogen (E2)-primed animal. Using a polyclonal antibody to these progesterone-dependent proteins (PDP) we have isolated a recombinant cDNA clone corresponding to the mRNA for PDP from a cDNA library prepared using poly(A+) RNA from the endometrium of P-treated E2-primed cats. Comparison of Western blots using the polyclonal antibody and epitope selected antibody demonstrated that the multiple molecular weight and isoelectric forms of the PDP are immunologically related and potentially products of the same gene. Northern analysis revealed that the mRNA for the PDP in the endometrium of P-treated E2-primed cats was 1.8 kilobases in length. Using slot blot analysis, we found that the PDP mRNA levels were low in the endometrium of ovariectomized animals and undetectable in E2-treated animals. With 1 day of P treatment the PDP mRNA levels were readily detectable and they peaked after 5 to 7 days of P treatment. No PDP mRNA was detectable in myometrium, oviduct, or ovary. Sequence analysis revealed that PDP had significant homology to human, rat, and mouse cathepsin L at the nucleotide (80%, 74%, and 73%, respectively) and amino acid (68%, 65%, and 63%, respectively) level. We suggest that PDP via its collagenolytic and elastolytic activities as a cathepsin L is responsible for preparing the endometrium for blastocyst implantation.     

Molecular cloning and characterization of prolactin-like protein C complementary deoxyribonucleic acid.

In this report, we describe the isolation and characterization of a full length cDNA clone for rat prolactin-like protein C (PLP-C) and describe the expression of PLP-C mRNA in the developing rat placenta. Nucleotide sequence analysis of the PLP-C cDNA clone predicted a mature protein of 238 amino acids, including a 30-amino acid signal sequence. The predicted PLP-C amino acid sequence contains seven cysteine residues, three tryptophan residues, and two putative N-linked glycosylation sites. Six of the cysteine residues in PLP-C are located in positions homologous to the cysteines of pituitary prolactin (PRL). Additional sequence similarities with pituitary PRL and other members of the rat placental PRL family are evident. The PLP-C gene was localized to rat chromosome 17. Northern blot analysis showed that the PLP-C cDNA clone specifically hybridized to a 1.0-kilobase mRNA. PLP-C mRNA was first detectable between days 13 and 14 of gestation, peaked by day 18 of gestation, and remained elevated until term. In situ hybridization analysis indicated that PLP-C mRNA was specifically expressed by spongiotrophoblast cells and some trophoblast giant cells in the junctional zone region of rat chorioallantoic placenta.     

Molecular cloning and expression of mouse placental lactogen I complementary deoxyribonucleic acid

The mouse midpregnancy lactogen or placental lactogen I (mPL-I) is encoded by a 1.0-kilobase mRNA that appears transiently during gestation, with maximal amounts accumulating in the placenta at day 10 of pregnancy. Several cDNA clones for mPL-I have been isolated from a lambda gt11 expression library constructed from day 10-placental RNA. The cDNA sequence indicates that mPL-I is synthesized as a 224 amino acid precursor, and is secreted as a 194 amino acid glycosylated hormone. The deduced amino acid sequence of mPL-I is highly homologous to the known members of the PRL family in the mouse, and hybridization analysis indicates that the mouse genome contains several mPL-I genes. Introduction of the mPL-I cDNA in an expression vector into cultured mouse cells results in the synthesis and secretion of glycosylated mPL-I protein that is recognized by anti-mPL-I antiserum and is biologically active.     

Cloned complementary deoxyribonucleic acid probes for untranslated messenger ribonucleic acid components of mouse sarcoma ascites cells

Mouse sarcoma ascites cells contain several abundant mRNA species that occur to a large extent in an untranslated state. RNA preparations enriched in these species were used as starting material to construct recombinant plasmids. Cloned plasmids bearing sequences homologous to four of the untranslated mRNA species were identified by translation of hybrid-selected material. These plasmids, as well as a recombinant plasmid derived from chick alpha-actin mRNA, were used as probes for the estimation of mRNA levels in polyribosomes and in small ribonucleoprotein (RNP) particles of the ascites cells. Considerable amounts of the mRNA molecules belonging to the untranslated species were present in polyribosomes as well as in mRNPs. The actin mRNA, on the other hand, was present almost exclusively in polyribosomes. The distributions obtained by the hybridization assay resembled those estimated by translation of the same RNA preparations in cell-free systems. This indicates that the mRNA molecules of a given species engaged in translation in the cells and those present as untranslated RNP particles are equally effective in cell-free translation systems.