Isolation and identification of a cDNA clone of rat placental lactogen II

The developing rat placenta expresses two placental lactogens at different stages of pregnancy: rat placental lactogen I from Days 11 to 13 of pregnancy and rat placental lactogen II (rPLII) from Day 12 to term. In this paper, we describe cDNA clones for rPLII, which have been isolated from a Day 18 rat placental cDNA library. The rPLII clones hybrid-select a mRNA which translates in vitro to a protein of 25,000 daltons. This protein is processed by dog pancreatic microsomes to a 22,000-dalton form, identical in size to rPLII isolated from pregnant rat serum. Both forms are precipitated by an anti-rPLII antiserum and an anti-ovine prolactin antiserum. The mRNA for rPLII is first expressed in Day 12 placenta and reaches a maximum at about Day 18 of pregnancy, in parallel with the appearance of the hormone in serum. Sequencing of the cDNA shows that, unlike human placental lactogen which is 85% homologous to human growth hormone at the amino acid level, rPLII is much more closely related to the prolactins. Thus, rPLII is 52% homologous to rat prolactin at the amino acid level, but only 34% related to rat growth hormone. This is the second placental lactogen to be fully characterized, and in the rat this hormone appears to have evolved by a route quite different from that which produced placental lactogen in humans.     

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.     

A new member of the prolactin-growth hormone gene family expressed in mouse placenta.

Mouse placenta has been found to contain an mRNA that encodes a previously unidentified member of the prolactin-growth hormone family. This 1.1-kb mRNA (designated PRP mRNA) was detected as a cDNA clone that hydridized to a cDNA clone of mouse proliferin, a recently described growth-associated placental protein related to prolactin. PRP mRNA levels are highest in the fetal part of the placenta and peak at day 12 of gestation, decreasing gradually until term. The 972-bp sequence of PRP mRNA, determined from two cDNA clones, encodes a protein of 244 amino acid residues that has a hydrophobic leader sequence. The protein encoded by PRP mRNA has significant homology to all of the members of the prolactin family, yet is different from each of them; it also differs from mouse placental lactogen. Nucleotide sequence homology is most extensive between PRP and proliferin mRNAs, particularly at their 5' ends, where they share 92 of the first 97 nucleotides.     

Molecular cloning and nucleotide sequence of cDNA encoding the rat kidney S-adenosylmethionine synthetase.

We previously reported the isolation of a cDNA encoding the liver-specific isozyme of rat S-adenosylmethionine synthetase from a lambda gt11 rat liver cDNA library. Using this cDNA as a probe, we have isolated and sequenced cDNA clones for the rat kidney S-adenosylmethionine synthetase (extrahepatic isoenzyme) from a lambda gt11 rat kidney cDNA library. The complete coding sequence of this enzyme mRNA was obtained from two overlapping cDNA clones. The amino acid sequence deduced from the cDNAs indicates that this enzyme contains 395 amino acids and has a molecular mass of 43,715 Da. The predicted amino acid sequence of this protein shares 85% similarity with that of rat liver S-adenosylmethionine synthetase. This result suggests that kidney and liver isoenzymes may have originated from a common ancestral gene. In addition, comparison of known S-adenosylmethionine synthetase sequences among different species also shows that these proteins have a high degree of similarity. The distribution of kidney- and liver-type S-adenosylmethionine synthetase mRNAs in kidney, liver, brain, and testis were examined by RNA blot hybridization analysis with probes specific for the respective mRNAs. A 3.4-kilobase (kb) mRNA species hybridizable with a probe for kidney S-adenosylmethionine synthetase was found in all tissues examined except for liver, while a 3.4-kb mRNA species hybridizable with a probe for liver S-adenosylmethionine synthetase was only present in the liver. The 3.4-kb kidney-type isozyme mRNA showed the same molecular size as the liver-type isozyme mRNA. Thus, kidney- and liver-type S-adenosylmethionine synthetase isozyme mRNAs were expressed in various tissues with different tissue specificities.     

Cloning and characterization of cDNA sequences corresponding to myosin light chains 1, 2, and 3, troponin-C, troponin-T, alpha-tropomyosin, and alpha-actin

A library of cDNA clones was constructed from adult rat skeletal muscle mRNA, from which a set of contractile protein clones was selected. These clones were identified by sequencing the cDNA inserts and comparing the derived amino acid sequences with published sequences of rabbit contractile proteins. In this manner, clones corresponding to myosin light chains 1, 2, and 3, troponin-C, troponin-T, alpha-tropomyosin, and alpha-actin were identified. A high degree of amino acid sequence conservation was found upon comparison of the rat and rabbit proteins. Using the cDNA clone panel, we analyzed the expression of abundant rat muscle mRNAs. We show that abundant rat muscle mRNAs can be classified into four developmentally regulated groups, based upon their expression at different stages of myogenesis. One class of mRNAs is expressed during all stages of muscle development. Since these mRNAs are also present in nonmuscle tissues, we conclude that they code for housekeeping proteins. The second class of mRNAs is present in both embryonic and adult muscle, while a third class of mRNAs is expressed only in adult muscle. A small number of mRNAs, which are present at greater levels in undifferentiated myoblasts than in adult muscle, comprise a fourth class. These results suggest the existence of at least four modes of gene control during myogenesis.     

Rat mammary gland fatty acid synthase: localization of the constituent domains and two functional polyadenylation/termination signals in the cDNA.

The rat fatty acid synthase (FAS) is active only as a dimer, although the eight component functions are contained in a single polypeptide chain. Using mRNA from lactating rat mammary glands a cDNA expression library was established. With the overlapping immunologically positive clones we have an 8.9kb cDNA sequence for rat FAS. In the 3'-nontranslated region of the rat FAS cDNA we find a prototype polyadenylation/termination signal and 779 nucleotides upstream, a mutated one. Both of these polyadenylation/termination signals are used and give rise to two equally abundant mRNA species which are coordinately regulated. In the derived amino acid sequence we could locate six of the eight component functions; their order is NH2- beta-ketoacyl synthase - acetyl/malonyl transferases -enoyl reductase - acyl carrier protein - thioesterase -COOH. Comparison of FAS from different sources shows that the primary sequence is conserved only for the active residues and the amino acids in their immediate vicinity.