RESUMO
The c-myc proto-oncogene is amplified and expressed at high levels in HL-60 cells, a cell line derived from a patient with acute promyelocytic leukemia. Upon induction to terminal maturation, expression of c-myc is greatly reduced. We have studied the level of gene expression at which the change in c-myc expression is controlled and the changes in chromatin configuration that accompany the repression of myc expression. We report here that the repression of myc expression with induced maturation is controlled at the level of transcription, and that reduced expression is accompanied by the loss of a single DNAse I hypersensitive site 0.9 kilobase pair upstream from the gene.
Assuntos
Diferenciação Celular , Cromatina/ultraestrutura , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Desoxirribonuclease I , Dimetil Sulfóxido/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proto-Oncogene Mas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Multiple isoenzymes of the Na+,K+-ATPase (alpha, alpha+, and alpha 3) have been identified by molecular cloning (Shull, G. E., J. Greeb, and J. B. Lingrel. 1986. Biochemistry. 25:8125-8132; and Schneider, J. W., R. W. Mercer, and E. J. Benz, Jr. 1987. Clin. Res. 35:585A. [Abstr.]). At least one of these, the alpha 3 chain, represents a novel form for which protein products and enzymatic activities are just beginning to be defined in rodents. We have recently demonstrated that expression of alpha 3 is largely confined to neuromuscular tissues of fetal and adult rats (Schneider, J. W., R. W. Mercer, M. Gilmore-Hebert, M. F. Utset, C. Lai, A. Greene, and E. J. Benz, Jr. 1988. Proc. Natl. Acad. Sci. USA. 85:284-288). We now report that certain human leukemia cell lines including HL60, HEL, and Molt 4 express mRNA for both alpha and alpha 3 isoforms of Na+,K+-ATPase; mRNA was not detected in several other cell lines, including K562 and U937; no cell lines expressed alpha+ mRNA. In uninduced HL60 cells, alpha 3 mRNA comprised 20-30% of total Na+,K+-ATPase mRNA. Furthermore, in HL60 and HEL cells, both alpha and alpha 3 mRNA declined after induction of maturation by DMSO, retinoic acid, or hemin. However, the reduction in alpha 3 mRNA was far more dramatic. alpha 3 mRNA virtually disappeared, but alpha mRNA declined by only approximately 50%. In contrast, when maturation of HL60 cells along the monocyte/macrophage lineage was induced by exposure to phorbol esters, alpha 3 mRNA remained abundant. Moreover, mRNA for the beta subunit of the Na+,K+-ATPase increased dramatically. Our results demonstrate that the alpha 3 isoform, formerly thought to be confined to neuromuscular tissues, is expressed in restricted lineages of hematopoietic origin. These leukemia cell lines should provide a useful model for analyzing regulation of the alpha 3 isoform gene and characterization of alpha 3 isoform activities.
Assuntos
Sistema Hematopoético/enzimologia , Isoenzimas/genética , ATPase Trocadora de Sódio-Potássio/genética , Animais , Northern Blotting , Linhagem Celular , DNA , Sondas de DNA , Regulação da Expressão Gênica , Humanos , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , RatosRESUMO
Messenger RNAs in eukaryotic cells exhibit a broad range of stabilities in vivo. Globin mRNA has a half life in excess of 50 h, but the half life of the c-myc oncogene mRNA is less than 20 min. Regulation of gene expression may be accomplished by a variety of mechanisms, including altering mRNA stability. We have examined the nuclear and cytoplasmic fractions of cells for factors affecting the metabolism of mRNA. Here we report that a HeLa whole-cell extract contains a factor that protects beta-globin mRNA from attack by RNases in a mouse erythroleukemia cell cytoplasmic extract. The factor is non-dialysable, inactivated by proteinase K and heat treatment, and resistant to RNase and DNase digestion. The HeLa cell factor resembles placental RNase inhibitor in that the mRNA-protecting activity is effective against RNase A and that treatment of the extract with N-ethylmaleimide completely destroys the protective activity. However, purified placental RNase inhibitor was unable to inhibit the RNase activity in the MELC cytoplasmic extract. These results suggest that the HeLa cell extract contains an RNase inhibitor (or inhibitors) with an activity or specificity that is distinct from that of placental RNase inhibitor.
Assuntos
Globinas/genética , Células HeLa/análise , RNA Mensageiro/metabolismo , Ribonucleases/antagonistas & inibidores , Linhagem Celular , Sistema Livre de Células , Etilmaleimida/farmacologia , Meia-Vida , Leucemia Eritroblástica Aguda/enzimologia , Proteínas de Neoplasias/metabolismo , Placenta/análise , Capuzes de RNA/metabolismoRESUMO
The rat carbonic anhydrase II gene was characterized and found to be approximately 15.5 kb in length and to contain 7 exons and 6 introns. All intron/exon junction and branch point sequences conform to consensus sequences, and the overall rat CA II genomic structure appears to be conserved upon comparison with mouse, human, and chicken CA II genes. The putative cis-acting elements within the analyzed 1014 bp 5' flanking region include: TATA box, 4 Sp1 binding sites, 2 AP2 sites and putative tissue-specific beta-globin-like repeat elements. A CpG island of approximately 800 bp was identified that begins about 600 bp upstream of exon 1 and extends about 200 bp into intron 1. In the 3' UTR, two polyadenylation signals (AATAAA) are present, the second of which is believed to be utilized. Northern blot analysis reveals that the 1.7 kb rat CA II mRNA is abundantly expressed in adult male brain and kidney, while negligible amounts are detected in heart and liver.
Assuntos
Anidrases Carbônicas/genética , Isoenzimas/genética , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Animais , Sequência de Bases , Anidrases Carbônicas/biossíntese , Anidrases Carbônicas/química , Galinhas , Sequência Conservada , Éxons , Biblioteca Genômica , Humanos , Íntrons , Isoenzimas/química , Masculino , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Transcrição GênicaRESUMO
A carbonic anhydrase II (CAII)-encoding cDNA clone was isolated from a rat brain lambda gt11 library. The 1459-bp cDNA codes for 260 amino acids with sequence similarity to mouse and human CAII and hybridizes to a single 1.7-kb mRNA.
Assuntos
Encéfalo/enzimologia , Anidrases Carbônicas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Anidrases Carbônicas/química , Clonagem Molecular , DNA/genética , Isoenzimas , Dados de Sequência Molecular , Ratos , Mapeamento por RestriçãoRESUMO
Ehlers-Danlos syndrome type IV (EDS IV) is characterized by variable changes in the skin, arterial fragility, bowel perforation, minimal joint involvement, and either autosomal recessive or autosomal dominant inheritance. The unifying biochemical abnormality is a deficiency of type III collagen; all patients studied thus far have shown a defect in either synthesis or in secretion of type III procollagen. We report on an adolescent boy who inherited EDS IV from his father and who developed a spontaneous subclavian artery aneurysm. In vitro studies of collagen production in dermal fibroblasts showed normal amounts of pro alpha 1 (III) messenger RNA and synthesis and secretion of nearly equal amounts of normal and of structurally abnormal pro alpha 1 (III) monomers. This patient is biochemically distinct from previous cases of EDS IV and is likely heterozygous for a mutation that results in an aberrant type III procollagen that is particularly susceptible to protease degradation.
Assuntos
Síndrome de Ehlers-Danlos/genética , Pró-Colágeno/genética , Adolescente , Aneurisma/genética , Células Cultivadas , Síndrome de Ehlers-Danlos/metabolismo , Genes Dominantes , Humanos , Masculino , Linhagem , Pró-Colágeno/metabolismo , Pele/metabolismoRESUMO
We present a patient with pansynostosis, hydrocephalus, seizures, extreme proptosis with luxation of the eyes out of the lids, apnea and airway obstruction, intestinal non-rotation, and severe developmental delay. His skeletal abnormalities include bilateral elbow ankylosis, radial head dislocation, and unilateral broad and deviated first toe. The phenotype of this patient is consistent with that previously reported in Pfeiffer syndrome type III, but is unusual for the lack of broad thumbs. Our patient most closely resembles the case described by Kerr et al. [1996: Am J Med Genet 66:138-143] as Pfeiffer syndrome type III with normal thumbs. Mutations in the genes for fibroblast growth factor receptors (FGFR) 1 and 2 have previously been seen in patients with Pfeiffer syndrome type I. The mutation identified in our patient, Ser351Cys in FGFR2, represents the first reported cause of Pfeiffer syndrome type III. An identical mutation was described once previously by Pulleyn et al., in a patient whose brief clinical description included cloverleaf skull, significant developmental delay, and normal hands and feet [Eur. J. Hum. Genet. 4: 283-291, 1996]. In our patient, previously performed single-strand conformation polymorphism analysis failed to detect a band shift; the mutation was identified only after independent sequence analysis.
Assuntos
Anormalidades Múltiplas/genética , Acrocefalossindactilia/genética , Substituição de Aminoácidos/genética , Mutação Puntual , Receptores Proteína Tirosina Quinases/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Acrocefalossindactilia/diagnóstico por imagem , Cisteína/genética , Cotovelo/anormalidades , Cotovelo/diagnóstico por imagem , Anormalidades do Olho/genética , Deformidades Congênitas do Pé/diagnóstico por imagem , Deformidades Congênitas do Pé/genética , Deformidades Congênitas da Mão/diagnóstico por imagem , Deformidades Congênitas da Mão/genética , Humanos , Recém-Nascido , Masculino , Fenótipo , Polimorfismo Conformacional de Fita Simples , Radiografia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Serina/genética , Translocação Genética , Cadeiras de RodasRESUMO
The term Baller-Gerold syndrome was coined by Cohen [1979: Birth Defects 15(5B): 13-63] to designate the phenotype of craniosynostosis and radial aplasia. It is thought to be a rare autosomal recessive condition, which, in some patients, presents with additional abnormalities, such as polymicrogyria, mental retardation or anal atresia. A phenotypic overlap of Baller-Gerold and Roberts-SC phocomelia syndrome was noted when a patient with bicoronal synostosis and bilateral radial hypoplasia was found to have premature centromere separation, a finding characteristic of Roberts syndrome [Huson et al.,1990: J Med Genet 27:371-375]. Other cases of presumed Baller-Gerold syndrome were rediagnosed as Fanconi pancytopenia, Rothmund-Thomson syndrome or VACTERL association. These reports led to a narrowed redefinition of Baller-Gerold syndrome based on the exclusion of cytogenetic and hematopoetic abnormalities and the absence of additional malformations in patients with craniosynostosis and preaxial upper limb abnormalities. Here we report on a patient with unilateral radial aplasia and bicoronal synostosis without additional malformations and without chromosome breakage, who fits this narrow definition of Baller-Gerold syndrome. We identified a novel TWIST gene mutation in this patient, a Glu181Stop mutation predicting a premature termination of the protein carboxy-terminal to the helix 2 domain. This report provides further evidence that Baller-Gerold is of heterogeneous cause, and a thorough evaluation is indicated to identify a possibly more specific diagnosis, including Saethre-Chotzen syndrome. This differential diagnosis is of particular importance, as it is an autosomal dominant trait. Therefore, the recurrence risk for parents of an affected child can be 50% if one parent carries the mutation, as opposed to the 25% recurrence risk for autosomal recessive inheritance. Offspring of the affected patient also have a 50% risk to inherit the mutation, while the risk to bear an affected offspring for an autosomal recessive trait is very low.
Assuntos
Craniossinostoses/genética , Heterogeneidade Genética , Mutação , Proteínas Nucleares , Rádio (Anatomia)/anormalidades , Fatores de Transcrição/genética , Adolescente , Adulto , Fácies , Feminino , Humanos , Recém-Nascido , Masculino , Síndrome , Proteína 1 Relacionada a TwistRESUMO
Confluent skin fibroblast cultures were prepared from 40 patients diagnosed with and surgically treated for an abdominal aortic aneurysm. An analysis of secreted type I and type III collagen in the media of these fibroblast preparations revealed reduced secretion of type III collagen from six patients. DNA sequence analysis of the entire coding domain of the pro alpha 1 (III) collagen mRNA in skin fibroblast RNA from these six patients revealed a C to T substitution at nucleotide 607 in one of the probands that would result in the replacement of a leucine residue with phenylalanine in the second position of the first tripeptide repeat in the triple-helical domain of type III collagen. Allele-specific hybridization analysis of genomic DNA from this proband and family members indicated that this non-glycine substitution probably contributed to the aneurysmal phenotype in this patient. No coding sequence mutations were found in the other five patients. It is clear from this study, therefore, that aberrant synthesis of type III collagen, as a consequence of both a coding sequence mutation and other factors contributing to reduced secretion of type III procollagen, will result in the development of an aortic aneurysm in a significant percentage of patients with this disease.
Assuntos
Aneurisma da Aorta Abdominal/genética , Colágeno/genética , Mutação Puntual , Idade de Início , Idoso , Aneurisma/epidemiologia , Aneurisma/genética , Aneurisma/metabolismo , Aneurisma/patologia , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/cirurgia , Arteriosclerose/metabolismo , Estudos de Coortes , Colágeno/biossíntese , Análise Mutacional de DNA , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Reação em Cadeia da Polimerase , Artéria Poplítea , Pró-Colágeno/metabolismo , Pele/metabolismoRESUMO
Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that predisposes the affected individual to develop characteristic tumors. These include CNS hemangioblastoma, retinal angiomas, endolymphatic sac tumors, pancreatic cysts and tumors, epididymal cystadenomas, pheochromocytomas, renal cysts, and clear-cell renal carcinoma. The VHL gene was localized to 3p25 and then isolated by Latif et al. (1). The gene contains three exons with an open reading frame of 852 nucleotides, which encode a predicted protein of 284 amino acids. The VHL protein is believed to have several functions. It is involved in transcription regulation through its inhibition of elongation by binding to the B and C subunits of elongin. Mutations of VHL allow the B and C subunits to bind with the A subunit. This complex then overcomes "pausing" of RNA polymerase during mRNA transcription (2,3). Several studies suggest that the VHL protein is also involved in regulation of hypoxia-inducible transcripts, particularly vascular endothelial growth factor (VEGF), by altering mRNA stability (4,5). Therefore, VHL gene mutations permit the overexpression of VEGF under normoxic conditions, which leads to the angiogenesis believed to be required for tumor growth. The VHL-elongin BC complex (VBC) also binds two other proteins-CUL2 and Rbx1-in a complex that has structural similarity to other E3 ubiquitin ligase complexes (6). Such complexes mediate the degradation of cell-cycle regulatory proteins.
Assuntos
Síndrome de Ehlers-Danlos/genética , Pró-Colágeno/genética , Criança , Síndrome de Ehlers-Danlos/classificação , Éxons/genética , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Masculino , Fenótipo , Mutação Puntual , Reação em Cadeia da Polimerase , Pró-Colágeno/deficiência , Splicing de RNA , RNA Mensageiro/genética , Pele/metabolismo , Pele/patologiaRESUMO
Patients with beta zero thalassemia arising from premature terminator codon mutations in the gene for beta globin do not produce beta globin protein; these individuals also exhibit a decreased amount of beta globin mRNA in their erythroid cells. The absence of beta globin protein is readily explained by the inability of the beta zero-39 mRNA to be translated. The decrease in beta globin mRNA has been attributed to either decreased cytoplasmic stability of the nontranslatable decreased cytoplasmic stability of the nontranslatable mRNA or to an undefined nuclear lesion. To compare directly the relative stabilities of normal and beta zero-39 thalassemic globin transcripts, we prepared normal and thalassemic beta globin pre-mRNAs and mRNAs using cloned DNA templates and the SP6 promoter-polymerase system. The stability of the transcripts was assessed by incubation in various cell-free extracts. Our results indicate that although the stabilities of the beta globin transcripts varied considerably from one extract to another the stabilities of the beta zero-39 thalassemic pre-mRNAs and mRNAs were equal to those of normal beta globin mRNAs in every extract tested.
Assuntos
Globinas/fisiologia , Talassemia/genética , Transcrição Gênica , Animais , Extratos Celulares/análise , Colódio , Citoplasma/metabolismo , Eletroforese em Gel de Ágar , Globinas/genética , Células HeLa/metabolismo , Humanos , Plasmídeos , Biossíntese de Proteínas , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Reticulócitos/análise , Reticulócitos/metabolismoRESUMO
Saethre-Chotzen syndrome is a relatively common craniosynostosis disorder with autosomal dominant inheritance. Mutations in the TWIST gene have been identified in patients with Saethre-Chotzen syndrome. The TWIST gene product is a transcription factor with DNA binding and helix-loop-helix domains. Numerous missense and nonsense mutations cluster in the functional domains, without any apparent mutational hot spot. Two novel point mutations and one novel polymorphism are included in this review. Large deletions including the TWIST gene have been identified in some patients with learning disabilities or mental retardation, which are not typically part of the Saethre-Chotzen syndrome. Comprehensive studies in patients with the clinical diagnosis of Saethre-Chotzen syndrome have demonstrated a TWIST gene abnormality in about 80%, up to 37% of which may be large deletions [Johnson et al., 1998]. The gene deletions and numerous nonsense mutations are suggestive of haploinsufficiency as the disease-causing mechanism. No genotype phenotype correlation was apparent.
Assuntos
Acrocefalossindactilia/genética , Mutação/genética , Proteínas Nucleares/genética , Fatores de Transcrição , Acrocefalossindactilia/diagnóstico , Humanos , Fenótipo , Polimorfismo Genético/genética , Proteína 1 Relacionada a TwistRESUMO
Human pro alpha 1 (I) and pro alpha 2 (I) procollagen chains have been separated on a large por (33 nm) reverse phase high performance liquid chromatography C18 column using an acetonitrile aqueous gradient containing 9 mM trifluoroacetic acid. The separation of the chains was evaluated by gel electrophoresis. The yield of the chromatography, based on radiolabel recovery, is equal to or better than 80%. Absorbance monitoring at 210 nm permits detection of less than 5 micrograms procollagen.
Assuntos
Pró-Colágeno/isolamento & purificação , Cromatografia Líquida de Alta Pressão , HumanosRESUMO
The synthesis of type III procollagen was examined in cultured fibroblasts from ten patients with type IV Ehlers-Danlos syndrome, a heritable disorder of connective tissue. With fibroblasts from nine patients, a decreased amount of labeled type III procollagen was recovered in the medium after the cells were incubated with radioactive amino acids for 24 h. The results were compatible with undefined defects in type III procollagen. The culture medium from one patient contained apparently normal amounts of type III procollagen after a 24-h labeling. However, the pro-alpha 1(III) chains from the medium of the patient's fibroblasts appeared as an abnormally broad band when examined by gel electrophoresis in sodium dodecyl sulfate. Analysis of fragments generated by vertebrate collagenase and cyanogen bromide located a structural defect between amino acid residues 555 and 775 in half of the alpha 1(III) chains. Most of the patient's type III procollagen was susceptible to digestion by pepsin or a mixture of chymotrypsin and trypsin at temperatures at which normal type III procollagen resisted digestion. Cyanogen bromide digestion of samples of the patient's skin revealed that the amount of type III was reduced more than 4-fold. The results support the hypothesis that both normal and structurally altered pro-alpha 1(III) chains are being incorporated into type III procollagen synthesized by the patient's fibroblasts and that type III procollagen molecules containing one, two, or three structurally altered pro-alpha 1(III) chains are rapidly degraded by proteinases in the tissues.
Assuntos
Síndrome de Ehlers-Danlos/metabolismo , Endopeptidases/metabolismo , Pró-Colágeno/biossíntese , Linhagem Celular , Quimotripsina/metabolismo , Brometo de Cianogênio/metabolismo , Eletroforese em Gel de Poliacrilamida , Fibroblastos/metabolismo , Humanos , Colagenase Microbiana/metabolismo , Mutação , Pepsina A/metabolismo , Fragmentos de Peptídeos/metabolismo , Pró-Colágeno/genética , Pró-Colágeno/metabolismo , Pele/metabolismo , Tripsina/metabolismoRESUMO
Human cDNA libraries were screened with a cDNA fragment presumably encoding the 3' terminus of a procollagen carboxyl propeptide not identifiable as types I, II, III, or IV by protein sequence or Northern blot hybridization. One clone contained a 1350-base pair insert coding in part for 55 uninterrupted Gly-X-Y triplets. Comparison with the amino acid composition of the COOH-terminal cyanogen bromide (CB) peptides of the alpha 1 and alpha 2 type V collagen chains showed similarity only to the alpha 2(V)CB fragment. To identify the NH2 terminus of the peptide designated by methionine, an additional isolate was sequenced and found to contain a Gly-Met-Pro triplet. Thirty-one amino acids from the NH2 terminus of the alpha 2(V)CB9 fragment were then determined by Edman degradation and found to be identical to those derived from the cDNA clone. The DNA sequence encoding part of the triple helical region establishes for the first time the partial structure of a type V collagen chain. Although comparison of residues 796-1020 of the alpha 2(V) collagenous region with alpha 1 (III), alpha 1(I), and alpha 2(I) shows strong conservation of charged positions, the latter three chains appear considerably more similar to each other than to alpha 2(V). A striking feature of the alpha 2(V) sequence between 918-944 is the absence of proline residues. In the analogous region of alpha 1(I) where this amino acid is also lacking, a flexible site in the rigid triple helical structure of type I collagen has been observed (Hofmann, H., Voss, T., Kuhn, K. and Engel, J. (1984) J. Mol. Biol. 172, 325-343).
Assuntos
Colágeno/análise , Sequência de Aminoácidos , Aminoácidos/análise , Sequência de Bases , Colágeno/genética , Brometo de Cianogênio/farmacologia , DNA/análise , Enzimas de Restrição do DNA/metabolismo , Humanos , Peso Molecular , Pró-Colágeno/genética , RNA Mensageiro/análiseRESUMO
The gamma-globin genes from a patient homozygous for a deletion form of hereditary persistence of fetal hemoglobin (HPFH-1) have been cloned and sequenced. The DNA sequence of the patient's gamma-globin genes corresponds to a previously identified sequence framework (chromosome A) with the exception of 10 base changes. Seven of these base changes can be attributed to normal allelic variation generated by small gene conversion events. The remaining three base changes are present in a 0.76 kb HindIII fragment containing a putative enhancer located 3' to the A gamma-globin gene. The same three base changes have also been described in the Seattle variant of nondeletion HPFH. We have analyzed 16 alleles from non-HPFH individuals and five alleles from individuals with nondeletion or deletion HPFH for the presence of these base changes by polymerase chain reaction amplification of cloned or chromosomal DNA and hybridization to allele-specific oligonucleotide probes. Although these base changes were found in an individual with HPFH-2, they were not found in the DNA from two patients with nondeletion HPFH. More importantly, all three base changes were detected in DNA from five non-HPFH individuals and appear to be common in blacks. We conclude that these base changes do not correlate with an HPFH phenotype and that the significant mutation in HPFH-1 is the deletion of over 100 kb of genomic DNA.
Assuntos
Hemoglobina Fetal/genética , Globinas/genética , Hemoglobinopatias/genética , Sequência de Bases , Deleção Cromossômica , Clonagem Molecular , Genes , Humanos , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Mapeamento por RestriçãoRESUMO
A lambda gt11 library constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats was screened for rat tropoelastin cDNAs. The first screen, utilizing a human tropoelastin cDNA clone, provided rat tropoelastin cDNAs spanning 2.3 kb of carboxy-terminal coding sequence and extended into the 3'-untranslated region. A subsequent screen using a 5' rat tropoelastin cDNA clone yielded clones extending into the amino-terminal signal sequence coding region. Sequence analysis of these clones has provided the complete derived amino acid sequence of rat tropoelastin and allowed alignment and comparison with published bovine cDNA sequence. While the overall structure of rat tropoelastin is similar to bovine sequence, numerous substitutions, deletions, and insertions demonstrated considerable heterogeneity between species. In particular, the pentapeptide repeat VPGVG, characteristic of all tropoelastins analyzed to date, is replaced in rat tropoelastin by a repeating pentapeptide, IPGVG. The hexapeptide repeat VGVAPG, the bovine elastin receptor binding peptide, is not encoded by rat tropoelastin cDNAs. Variations in coding sequence between rat tropoelastin cDNA clones were also found which may represent mRNA heterogeneity produced by alternative splicing of the rat tropoelastin pre-mRNA.
Assuntos
RNA Mensageiro/genética , Tropoelastina/genética , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Aorta/metabolismo , Sequência de Bases , Bovinos , Clonagem Molecular/métodos , DNA/genética , Biblioteca Gênica , Dados de Sequência Molecular , Músculo Liso Vascular/metabolismo , Poli A/genética , RNA/genética , Ratos , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
A Sephadex G-50 medium (Pharmacia) buffer-exchange column has been developed for rapidly changing the medium for carcinoembryonic antigen from perchloric acid to acetate buffer. Analytical recovery of the extracted antigen exceeded 95%. Plasma for analysis can be so prepared and samples ready for analysis within an hour. The technique also results in uniform assay volume, thus eliminating this variable from the assay.