Hyphantria cunea ferritin heavy chain homologue: cDNA sequence and mRNA expression

We have sequenced a cDNA clone encoding a 26-kDa ferritin subunit, which was heavy chain homologue (HCH), in fall webworm, Hyphantria cunea. The HCH cDNA was obtained from the screening of a cDNA library using a PCR product. H. cunea ferritin is composed of 221 amino acid residues and their calculated mass is 26,160 Da. The protein contains the conserved motifs for the ferroxidase center typical for heavy chains of vertebrate ferritin. The iron-responsive element sequence with a predicted stem-loop structure is present in the 5'-untranslated region of ferritin HCH mRNA. The sequence alignment of ferritin HCH shows 68.9 and 68.7% identity with Galleria mellonella HCH (26 kDa ferritin) and Manduca sexta HCH, respectively. While G type insect ferritin vertebrate light chain homologue (LCH) is distantly related to H. cunea ferritin HCH (17.2-20.8%), the Northern blot analysis revealed that H. cunea ferritin HCH was ubiquitously expressed in various tissues and all developmental stages. The ferritin expression of midgut is more responsive to iron-fed, compared to fat body in H. cunea.     

H3 and H4 histone cDNA sequences from Xenopus: a sequence comparison of H4 genes

Ovarian poly (A) + RNA from Xenopus laevis and Xenopus borealis was used to construct two cDNA libraries which were screened for histone sequences. cDNA clones to H4 mRNA were obtained from both species and an H3 cDNA clone from Xenopus laevis. The complete DNA sequences of these clones have been determined and are presented. These new sequences are compared with other H3 and H4 DNA sequences both in the coding and 3' noncoding regions. We find that there is considerable non-random codon usage in ten H4 genes. In addition there are some sequence similarities in the 3' noncoding regions of H3 and H4 genes.     

Human liver glutathione S-transferases: complete primary sequence of an Ha subunit cDNA

Multiple human liver GSH S-transferases (GST) with overlapping substrate specificities may be essential to their multiple roles in xenobiotics metabolism, drug biotransformation, and protection against peroxidative damage. Human liver GSTs are composed of at least two classes of subunits, Ha (Mr = 26,000) and Hb (Mr = 27,500). Immunological cross-reactivity and nucleic acid hybridization studies revealed a close relationship between the human Ha subunit and rat Ya, Yc subunits and their cDNAs. We have determined the nucleotide sequence of the Ha subunit 1 cDNA, pGTH1. The alignments of its coding sequence with the rat Ya and Yc cDNAs indicate that they are approximately 80% identical base-for-base without any deletion or insertion. Regions of sequence homology (greater than 50%) have also been found between pGTH1 and a corn GST cDNA and rat GST cDNAs of the Yb and Yp subunits. Among the 62 highly conserved amino acid residues of the rat GST supergene family, 56 of them are preserved in the Ha subunit 1 coding sequences. Comparison of amino-acid replacement mutations in these coding sequences revealed that the percentage divergence between the rat Ya and Yc genes is more than that between the Ha and Ya or Ha and Yc genes.     

Hamster estrogen receptor cDNA: cloning and mRNA expression

Estrogen-induced hamster kidney tumor model serves as a useful model to study the biochemical and molecular mechanisms of hormonal carcinogenesis. In this model, we have demonstrated an increased expression of estrogen receptor mRNA and protein in estrogen-treated kidneys and in estrogen-induced tumors. The sequence information for hamster estrogen receptor gene is not known and has been investigated in this study. A hamster uterus cDNA library was constructed and the 5'-region of the hamster estrogen receptor cDNA cloned from this library using polymerase chain reaction (PCR) methodology. Additionally, hamster kidney polyadenylated RNA was reverse transcribed and PCR amplified using primers that were designed based on maximum homology between mouse, rat and human estrogen receptor cDNAs. These PCR amplified fragments were cloned into plasmid vectors and clones with the expected size of the insert subjected to Southern blot analysis using human estrogen receptor cDNA as a probe. The positive clones on Southern blot analysis and the PCR amplified products from these clones were subjected to DNA sequence analysis. Using this strategy, a full length, 1978 bp hamster estrogen receptor cDNA has been cloned which shows 87% homology with human, 90% with rat and 91% with mouse estrogen receptor cDNA. The deduced amino acid shares 88% homology with human, and 93% with rat and mouse estrogen receptors. Hamster estrogen receptor domain C (DNA binding domain) shows a 100% homology with a similar domain from mouse, rat, human, pig, sheep, horse and chicken estrogen receptor (Genebank reference ID: AF 181077).     

The nucleotide sequence of a rat liver glutathione S-transferase subunit cDNA clone

We have determined the nucleotide sequence of a cloned cDNA derived from liver poly(A) RNA of pentobarbital-treated rats encoding a glutathione S-transferase subunit. This cDNA clone pGTR261 contains one open reading frame of 222 amino acids, a complete 3' noncoding region, and 63 nucleotides in the 5' noncoding region. The cloned DNA hybridizes to rat poly(A) RNA in a tissue-specific fashion, with strong signals to liver and kidney poly(A) RNA(s) of approximately 1100 and approximately 1400 nucleotides in size but little or no hybridization to poly(A) RNAs from heart, lung, seminal vesicles, spleen, or testis under stringent conditions. Our sequence covers the cDNA sequence of pGST94 which contains a partial coding sequence for a liver glutathione S-transferase subunit of Ya size. Comparison of sequences with our earlier clone pGTR112 suggests that there are at least two mRNA species coding for two different subunits of the Ya (Mr = 25,600) subunit family with very limited amino acid substitutions mainly of conserved polarity. The divergent 3' noncoding sequences should be useful molecular probes in differentiating these two different but otherwise very similar subunits in induction and genomic structure analyses. Our results suggest that tissue-specific expression of the glutathione S-transferase subunits represented by the sequences of pGTR261 and pGTR112 may occur at or prior to the level of RNA processing.     

Differential expression of Atlantic salmon thyrotropin β subunit mRNA and its cDNA sequence

To study the regulation of the thyroid system, an Atlantic salmon Salmo salar cDNA clone was isolated for thyroid stimulating hormone (TSH) β subunit gene. A cDNA (866 bp) was isolated from an adult Atlantic salmon pituitary cDNA library, this clone was sequenced and shown to be highly conserved when compared to other teleost β TSH subunit sequences. The cDNA was used as a probe for Northern blot analysis of total pituitary RNA from the different life cycle stages of Atlantic salmon. Northern blot analysis demonstrated that β TSH mRNA is expressed at all life cycle stages studied, including parr, smolt, immature fish at sea and sexually mature male fish. Densitometry of Northern blots showed that sexually mature male salmon had low levels of salmon β TSH mRNA compared to non-mature fish. Stunts, fish performing poorly in salt water, were shown to have elevated levels of β TSH mRNA when compared to healthy fish.     

Dexamethasone and estrogen regulate Xenopus laevis albumin mRNA levels

The regulation of albumin mRNA levels by steroid hormones was examined in a primary Xenopus liver culture system. In the absence of exogenous steroid hormone, albumin mRNA levels declined rapidly in culture. Dexamethasone is required for preservation of albumin mRNA levels in culture and effects a greater than 10 fold induction of albumin mRNA in cultures maintained in steroid hormone-free medium. Estrogen can override the effect of dexamethasone and elicits a decline of greater than 80% in albumin mRNA levels. Our cDNA clones of the mRNAs encoding the two 74,000 dalton and one 72,000 dalton cellular albumins allowed us to show that all three albumin mRNAs were down regulated by estrogen.     

cDNA sequence of the human integrin beta 5 subunit

A novel integrin receptor involved in cell adhesion to the matrix protein vitronectin has recently been described from a human lung epithelial-derived cell line (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69). This receptor has an alpha subunit that appears identical to the alpha v of the vitronectin receptor alpha v beta 3 expressed in melanoma and endothelial cells, but is complexed with a distinct beta subunit, beta 5. cDNA clones coding for beta 5 have been isolated and used to determine the mRNA and amino acid sequence of this new subunit. A 3.3-kilobase mRNA was found to code for a mature protein of 775 amino acid residues with a hydrophobic leader sequence of 24 amino acids. A 56% identity was found between the beta 5 and beta 3 protein sequences, making them the most closely related of the integrin beta subunits. Polymerase chain reaction abundance analysis revealed that alpha v and beta 5 mRNAs were found in seven very different cell lines, compared with beta 3 mRNA which was found in only three of the them, indicating that this new integrin receptor may be widely distributed.     

Dendritic translocation of the rat ferritin H chain mRNA

To elucidate the mechanism regulating the selective transport of mRNAs to synaptic sites, we compared the synaptosomal mRNAs with those from the forebrain using the differential display method. The ferritin H chain mRNA was found to be highly enriched in the synaptosomes. In situ hybridization for the ferritin H chain mRNA in the cultured dissociated neurons and in the hippocampal brain slices demonstrated its existence in the dendritic region. These data clearly indicate the selective translocation of the ferritin H chain mRNA into the dendrites and suggested the local expression of ferritin at the synapse.     

Isolation and partial amino acid sequence of three subunit species of porcine spleen ferritin: evidence of multiple H subunits

A partial amino acid sequence for three different subunits of the iron storage protein, ferritin, has been determined. Ferritin (Mr approximately 480,000) was isolated from porcine spleen and dissociated into its component subunits (Mr approximately 20,000). The subunits, in turn, were separated into three fractions by reversed-phase HPLC. The fractions appeared to be of equal size by sedimentation velocity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and size-exclusion chromatography in 6 M guanidinium chloride. All three fractions were shown to be monomeric and to have no covalently attached carbohydrate (J. F. Collawn et al. (1984) Arch. Biochem. Biophys. 233, 260-266). Determination of the amino acid sequence of the C-terminal 70-80 residues from each of the fractions demonstrated three different sequences. Comparison with human liver H and L subunit sequences indicates that two of the porcine ferritin subunits are H-type subunits and one is an L-type subunit. Application of the Chou-Fasman algorithm on the three partial sequences suggests that these respective regions from each of the three subunits would probably adopt the same conformation.     

Cloning and sequence analysis of a cDNA for a rat liver glutathione S-transferase Yb subunit.

We have isolated a Yb-subunit cDNA clone from a GSH S-transferase (GST) cDNA library made from rat liver polysomal poly(A) RNAs. Sequence analysis of one of these cDNA, pGTR200, revealed an open reading frame of 218 amino acids of Mr = 25,915. The deduced sequence is in agreement with the 19 NH2-terminal residues for GST-A. The sequence of pGTR200 differs from another Yb cDNA, pGTA/C44 by four nucleotides and two amino acids in the coding region, thus revealing sequence microheterogeneity. The cDNA insert in pGTR200 also contains 36 nucleotides in the 5' noncoding region and a complete 3' noncoding region. The Yb subunit cDNA shares very limited homology with those of the Ya or Yc cDNAs, but has relatively higher sequence homology to the placental subunit Yp clone pGP5. The mRNA of pGTR200 is not expressed abundantly in rat hearts and seminal vesicles. Therefore, the GST subunit sequence of pGTR200 probably represents a basic Yb subunit. Genomic DNA hybridization patterns showed a complexity consistent with having a multigene family for Yb subunits. Comparison of the amino acid sequences of the Ya, Yb, Yc, and Yp subunits revealed significant conservation of amino acids (approximately 29%) throughout the coding sequences. These results indicate that the rat GSTs are products of at least four different genes that may constitute a supergene family.     

Nucleotide sequence of cloned cDNA for pro-opiomelanocortin in the amphibian Xenopus laevis

The function of alpha-melanocyte-stimulating hormone (alpha-MSH) is not known in mammals. It is well-established in the amphibian Xenopus laevis in which alpha-MSH mediates the process of adaptation to a dark background. The amino acid sequence of this hormone is, however, not known in amphibians. In order to determine the primary structure of the precursor protein for alpha-MSH, which in mammals has been called pro-opiomelancortin (POMC), we constructed a cDNA library from Xenopus pituitary mRNA. A pool of synthetic oligodeoxyribonucleotide tetradecamers corresponding to part of the mammalian alpha-MSH sequence was used to screen the library. The nucleotide sequence of a 1050-base pair hybridization-positive cDNA clone was determined and the deduced amino acid sequence of Xenopus POMC revealed the sequences of Xenopus gamma-MSH, alpha-MSH, corticotropin-like intermediate lobe peptide, beta-MSH, and beta-endorphin. Interestingly, the N-terminal amino acid of Xenopus alpha-MSH, which is N alpha-acetylated in the biologically active form of the hormone, is different from that of mammalian alpha-MSH. The distribution of the bioactive domains within Xenopus POMC is remarkably similar to that in other known POMC molecules and as in mammals the domains in the amphibian prohormone are flanked on both sides by pairs of basic amino acids.