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Query: UNIPROT:P06889 (Mol)
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We have isolated and cloned the full length cDNA for mouse GH-releasing hormone (mGRH) from mouse hypothalamus using a recently described strategy involving the polymerase chain reaction technique (PCR). Degenerate oligonucleotide primers were selected based on short (six amino acids) conserved regions in the human and rat GRH peptides that would recognize DNA sequences encoding similar amino acids regardless of codon usage. Primer-extended cDNA was amplified by PCR on cDNA templates prepared by reverse transcribing total mouse hypothalamic RNA. After cloning and sequencing the initial product, the 3' and 5' ends of mGRH were generated using a separate PCR strategy (RACE protocol). The mGRH cDNA encodes a 103-amino acid reading frame, structurally similar to the human and rat GRH genes, containing a signal sequence, a 42-residue GRH peptide, and a 31-residue C-terminal region. Although the structures of mouse and rat GRH are highly conserved in the signal peptide and C-terminal region, there is considerable diversity in the GRH region, which exhibits nearly comparable homology with the rat (68%) and human (62%) structures. Differences between mouse and rat GRH were also found in the amino acid cleavage sites at the 5' and 3' ends of the mature peptide and at the polyadenylation signal.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1989 Oct
PMID:Cloning and characterization of mouse growth hormone-releasing hormone (GRH) complementary DNA: increased GRH messenger RNA levels in the growth hormone-deficient lit/lit mouse. 248 13

We have isolated and sequenced a cDNA and region of genomic DNA that encode HCCP12, a major protein found in flexible cuticles of all three metamorphic stages of the giant silkmoth, Hyalophora cecropia. The gene for HCCP12 contains two introns with the first intron interrupting the signal peptide. The 5' flanking region contains 478 base pairs (bp) with about 95% identity to the prototype of a common Cecropia insertion element. The RACE procedure was used to produce cDNAs corresponding to the 5' ends of mRNAs for HCCP12 isolated from larval, pupal and pharate adult epidermal cells. Sequences from 21 cloned cDNAs were almost identical. It was thus concluded that a single gene and a single promoter (as evidenced by a single transcriptional start site) for HCCP12 are used in all three metamorphic stages. Hence, neither stage-specific gene sets nor stage-specific promoters are essential for metamorphic transitions of cuticular protein gene expression.
Insect Biochem Mol Biol 1994 Dec
PMID:Identification of the cDNA, gene and promoter for a major protein from flexible cuticles of the giant silkmoth Hyalophora cecropia. 753 19

To explore the role of the matrix metalloproteinase matrilysin (MAT) in normal tissue remodeling, we cloned the murine homologue of MAT from postpartum uterus using RACE polymerase chain reaction and examined its pattern of expression in embryonic, neonatal, and adult mice. The murine coding sequence and the corresponding predicted protein sequence were found to be 75% and 70% identical to the human sequences, respectively, and organization of the six exons comprising the gene is similar to the human gene. Northern analysis and in situ hybridization revealed that MAT is expressed in the normal cycling, pregnant, and postpartum uterus, with levels of expression highest in the involuting uterus at early time points (6 h to 1.5 days postpartum). The mRNA was confined to epithelial cells lining the lumen and some glandular structures. High constitutive levels of MAT transcripts were also detected in the small intestine, where expression was localized to the epithelial Paneth cells at the base of the crypts. Similarly, MAT expression was found in epithelial cells of the efferent ducts, in the initial segment and cauda of the epididymis, and in an extra-hepatic branch of the bile duct. MAT transcripts were detectable only by reverse transcription-polymerase chain reaction in the colon, kidney, lung, skeletal muscle, skin, stomach, juvenile uterus, and normal, lactating, and involuting mammary gland, as was expression primarily late in embryogenesis. Analysis of MAT expression during postnatal development indicated that although MAT is expressed in the juvenile small intestine and reproductive organs, the accumulation of significant levels of MAT mRNA appears to correlate with organ maturation. These results show that MAT expression is restricted to specific organs in the mouse, where the mRNA is produced exclusively by epithelial cells, and suggest that in addition to matrix degradation and remodeling, MAT may play an important role in the differentiated function of these organs.
Mol Biol Cell 1995 Jul
PMID:The metalloproteinase matrilysin is preferentially expressed by epithelial cells in a tissue-restricted pattern in the mouse. 757 99

Cyclins are a complex group of proteins involved in regulation of the eukaryotic cell division cycle via their interaction with cyclin dependent kinases (Cdks). Cyclin gene sequences have been cloned from a number of plant species, including alfalfa, but the diversity of these genes suggests that there are many plant cyclins which have yet to be characterized. A RACE-PCR strategy has been adopted for cloning cyclin gene sequences expressed during direct somatic embryogenesis in alfalfa. RT-PCR with nested degenerate primers was used to amplify the highly conserved "cyclin box" region of a novel A-like cyclin mRNA sequence expressed after induction of somatic embryogenesis. The sequence of this PCR product was used to design primers for 5'- and 3'-RACE protocols. 5'-RACE using a modified SLIC (single strand ligation to single stranded cDNA) procedure revealed considerable sequence heterogeneity in the N-terminal region of the coding sequence with several closely related sequences apparent. Conventional 3'-RACE generated a single cyclin sequence. The complete coding sequence of one member of this A-like cyclin subgroup has been obtained by this RACE strategy and confirmed by PCR amplification and sequencing of alfalfa genomic DNA.
Cell Mol Biol (Noisy-le-grand) 1995 Jul
PMID:Cloning novel alfalfa cyclin sequences--a RACE-PCR approach. 758 Aug 50

A new member of the S gene family, SLR3 (S-Locus Related 3), was identified in Brassica oleracea. This gene had a novel pattern of expression compared with previously described members of the family, being expressed in petals, sepals and vegetative apices, in addition to stigmas and anthers. Moreover, use of SLR3-derived probes in RNA blot and RACE-PCR (rapid amplification of cDNA ends-polymerase chain reaction) experiments has identified transcripts of genes closely related to SLR3 in leaves, cotyledons and, at high levels in developing anthers. SLR3 is not linked to the S locus but is linked to two or three closely related genes. Sequence analysis of the SLR3 gene indicates that it is derived from an ancestral receptor kinase gene that has been modified by a series of deletion events. As a result of these modifications, SLR3 is predicted to encode a secreted glycoprotein lacking both transmembrane and kinase domains. The putative SLR3 protein differs from the products of most other S gene family members in that several of the highly conserved cysteines have been lost. Within the S gene family, modification of receptor kinase genes by deletion may represent a general mechanism for the generation of genes encoding secreted glycoproteins.
Mol Gen Genet 1995 Jul 28
PMID:SLR3: a modified receptor kinase gene that has been adapted to encode a putative secreted glycoprotein similar to the S locus glycoprotein. 765 38

Relaxin is a peptide hormone which consists of two polypeptide chains that are synthesized as a B-chain/C-peptide/A-chain precursor. We have used the polymerase chain reaction (PCR) to isolate and clone a relaxin-like cDNA from sheep placental RNA. This cDNA and two sheep genomic clones were characterised by nucleotide sequencing. A comparison of the sheep nucleotide sequence with exon II of pig relaxin revealed homology of 72%. The sheep sequence had numerous stop codons in the region corresponding to the C-peptide. Therefore, there is no open reading frame which would include the C-peptide and A-chain regions. Analysis of several animals indicates that the stop codons are not due to an allelic polymorphism and Southern blot analysis of genomic DNA reveals the presence of a single copy gene. The 5' RACE PCR protocol was used to obtain sequence information for the 5' relaxin-like RNA. This analysis reveals that unprocessed precursor RNA is the predominant RNA species in placenta. A small proportion of clones was isolated which contained novel 5' sequences. These sequences mostly appear to be generated from repetitive DNA elements upstream of exon II. No relaxin-like exon I sequence which encodes the B-chain was found after an extensive search of the 5' RACE PCR products. Therefore, this relaxin-like gene does not produce an RNA species in ovary, placenta or endometrial tissue which could give rise to a functional sheep relaxin hormone.
Mol Cell Endocrinol 1993 Feb
PMID:A single-copy relaxin-like gene sequence is present in sheep. 768 20

Human pulmonary surfactant protein A (SP-A) is encoded by two genes, SP-A1 and SP-A2. Reports from our laboratory and other investigations have shown heterogeneity in both genes within three regions (the 5' untranslated [5' UT], the coding, and the 3' untranslated [3' UT] regions). To more fully examine the variability in these regions and characterize the transcription start site in each gene, we used primer extension and 5' RACE to clone and then sequence cDNA clones from two individuals. These cDNAs extended from the transcription start site to approximately 40% of the 3' UT segment. The in vitro translatability of selected cDNAs was also tested. After analysis of our data, we found that: (1) the 5' UT of SP-A genes contains four (A, B, C, D for SP-A1) or three (A, B, D for SP-A2) untranslated exons, three of which (A, B, D) vary in length, and one of which (C) is new; (2) these exons are alternatively spliced and the major splice patterns as well as their relative frequency vary between the two genes (the major pattern for SP-A1 is AD'[81%] and the major patterns for SP-A2 are ABD [44%] and ABD'[49%]); (3) the SP-A1 gene uses three transcription start sites with equal frequency, whereas the SP-A2 gene uses only one; (4) splicing variability occurs among alleles and among individuals; (5) three previously undescribed alleles exist for the SP-A1 gene (6A2, 6A3, 6A4) and two for the SP-A2 gene (1A1, 1A2); and (6) a core group of 10 invariant nucleotides and four invariant amino acids can be used to discriminate between SP-A1 and SP-A2 alleles.
Am J Respir Cell Mol Biol 1995 Jan
PMID:5' splicing and allelic variants of the human pulmonary surfactant protein A genes. 781 73

Brain-derived neurotrophic factor (BDNF) enhances the survival of dopaminergic neurons and protects them from neurotoxins in vitro. This trophic factor might thus be of therapeutic value for the treatment of Parkinsonian syndromes. The rat BDNF gene consists of several upstream noncoding exons that are alternatively spliced to a common coding exon. To investigate BDNF 5' exons expressed in the adult rat brain, we subjected RNA from cerebellum to 5'-RACE analysis and compared the resulting clones to previously reported 5' exon sequences from rat brain and hippocampus. In addition to known 5' exons, we isolated a BDNF transcript with a novel 5' sequence representing yet another alternate upstream exon in this gene. Quantitative PCR analysis of BDNF mRNAs containing each of the five upstream exons indicated that each of the alternate transcripts is most abundant in the hippocampus, intermediate in the substantia nigra and cerebellum and least abundant in the striatum. However, the magnitude of these differences in expression varied considerably suggesting that BDNF gene transcription in the mature brain is regulated by alternate promoters that are differentially active across regions.
Brain Res Mol Brain Res 1994 Oct
PMID:Alternate 5' exons in the rat brain-derived neurotrophic factor gene: differential patterns of expression across brain regions. 785 51

The polychaete worm Ctenodrilus serratus was surveyed for the presence of HOM/HOX and engrailed-type homeobox genes using PCR with degenerate primers. Sixteen unique homeobox fragments were found in surveys of genomic and cDNA with three different primer sets. For three fragments, RACE was employed to obtain additional homeobox sequence and the 3' flanking region. Nine HOM/HOX-type fragments were identified, including putative representatives of the Hox1/lab, Hox2/pb, Hox3, Hox4/Dfd, and Antp/Lox5 cognate groups. Two additional Antp-like fragments could not be assigned specific orthology. Presence of an ortholog of leech Lox2 in addition to a Ubx/abdA-like gene suggests that independent duplications of a single precursor occurred in the annelid and arthropod lineages. No representative of the Hox9/AbdB group was identified. Our results are consistent with a hypothesis of a single HOM/HOX cluster in Ctenodrilus as extensive as that seen in strongly tagmatized arthropods, suggesting that the primitive role of these genes even in overtly metameric animals was something other than specification of overt segmental differentiation. The primers used also detected representatives of six other homeobox classes or families: Xlox (XlHbox8/HTr-A2), Ovx (Chox7), caudal, Prh (proline-rich homeobox), NEC (ceh-9/Tghbox5), and engrailed.
Mol Phylogenet Evol 1994 Jun
PMID:A PCR-based survey of homeobox genes in Ctenodrilus serratus (Annelida: Polychaeta). 791 7

Polyphenol oxidase (PPO) was purified to homogeneity from Sultana grapes yielding a single protein with an apparent molecular mass of 40 kDa as determined by SDS-PAGE. A degenerate oligonucleotide primer based on the N-terminal amino acid sequence of this purified 40 kDa grape PPO protein was used to amplify a 1650 bp cDNA clone (GPO1M) by 3' rapid amplification of cDNA ends (3'-RACE). GPO1M hybridized to a single 2.2 kb transcript from grape berry mRNA indicating the presence of further upstream sequence which was cloned using 5'-RACE PCR. The complete 1990 bp cDNA (GPO1) encodes a 67 kDa protein consisting of a 10.6 kDa chloroplast transit peptide, a 40.5 kDa catalytic unit containing two copper-binding regions and a 16.2 kDa C-terminal extension. Southern analysis suggested the presence of only one PPO gene in grapevine. High levels of gene expression were found in young developing berries, leaves and roots, but there was little expression in mature tissues. Biogenesis of PPO in grapevine tissues, appears to involve synthesis of a 67 kDa precursor protein which is imported into the chloroplast and processed to remove a 10.6 kDa chloroplast transit peptide from the N-terminus and a 16.2 kDa peptide of unknown function from the C-terminus.
Plant Mol Biol 1994 Oct
PMID:Molecular cloning and characterisation of grape berry polyphenol oxidase. 794 97


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