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1.
E F Vanin  M G Farace  R Gambari  A Fantoni 《Gene》1981,16(1-3):141-148
We have determined the nucleotide sequence of two cloned cDNAs corresponding to the mRNA of mouse embryonic y2 globin. The combined overlapping sequences span a total of 480 bp, beginning at the codon corresponding to amino acido residue 21 and extending to the AATAAA sequence in the 3' untranslated region. Therefore, when the amino acid sequence encoded by the cDNA is combined with the available amino acid sequence, a complete y2 protein sequence can be obtained. Comparisons, at the nucleotide level, between the known beta- and beta-like globin sequences and the y2 sequence show that the embryonic, fetal-adult duplication occurred approx. 160 million years (MY) ago and that the embryonic-fetal duplication occurred approx. 100 MY ago.  相似文献   

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The construction of a double-stranded cDNA library using rat prostatic poly(A)RNA and pBR322/kappa 1776 system and the isolation of three prostatic binding protein (PBP) cDNA clones are described. These cDNA clones were characterized and identified by in situ hybridization, mRNA selection-translation and immuno-precipitation as coding for the three subunit components, C1, C2, and C3, of PBP. These clones were used in hybridization experiments with prostatic poly(A)RNA to determine the effect of testosterone on the levels of PBP-mRNA. The results showed that synthesis of these mRNAs varied in response to either androgen withdrawal or replacement. Accumulation of PBP-mRNAs coding for C2 and C3 components occurred 1 hr after androgen administration to castrated rat, whereas the mRNA coding for the C1 component did not appear until 4 hr after androgen replacement. Quantitation of PBP-mRNA sequences in nuclear and polysomal poly(A)RNAs showed that they did not vary coordinately in response to androgen withdrawal. These results indicate differential regulation of PBP genes and suggest possible multiple levels of androgen control of PBP synthesis.  相似文献   

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The 3' terminal nucleotide sequence of two clones containing DNA complementary to mu chain mRNA of IgM-secreting cells has been determined. The sequence shows a termination codon (UGA) adjacent to the terminal tyrosine codon for the secreted protein and a 3' non-coding region of at least 106 bases. The primary translation product of this mu chain mRNA seems to terminate at the tyrosine of the secreted protein.  相似文献   

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Cloned fragments of mouse DNA have been screened for the presence of long polypyrimidine/polypurine segments. The polypyrimidine portion of one such segment (about 200 nucleotides in length) has been isolated by acidic depurination of the entire cloned fragment and plasmid vector followed by selective precipitation and 5'-32P labeling. This polypyrimidine has been used to demonstrate a new procedure for sequencing. Covalent modification of thymine with a water-soluble carbodiimide, or cytosine with glutaric anhydride, at low levels blocked the action of snake venom exonuclease. After deblocking, separation of the products of digestion by polyacrylamide gel electrophoresis yields a sequence ladder which can be used to determine the position of C and T residues as in other sequencing methods. A sequence of 72 residues adjacent to the 5' end has been established, consisting principally of the repeating tetranucleotide (CCTT)n. A low ratio of endonuclease to exonuclease is essential for application of this method to sequences of this size. Accordingly, a very sensitive modification of a fluorometric endonuclease assay was developed and used to optimize pH and Mg2+ conditions to favor exonuclease activity over the accompanying endonuclease activity. The results clearly indicate that long polypyrimidine tracts can be efficiently prepared and their sequences determined with this method using commercially available exonuclease preparations without additional purification.  相似文献   

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The amino acid sequence of the bovine mitochondrial nicotinamide nucleotide transhydrogenase was recently deduced from isolated cDNAs and reported [Yamaguchi, M., Hatefi, Y., Trach, K., and Hoch, J.A. (1988) J. Biol. Chem. 263, 2761-2767]. The cDNAs lacked the N-terminal coding region, however, and the 8 N-terminal residues were determined by protein sequencing. In the present study, the nucleotide sequence of the 5' upstream region was determined by dideoxynucleotide sequencing of the transhydrogenase messenger RNA, and amino acid sequences of the N-terminal region and the signal peptide of the enzyme were deduced from the nucleotide sequence. The N-terminal sequence of the enzyme as deduced from the mRNA sequence is the same as that determined by protein sequencing, with one difference. Protein sequencing showed Ser as the N-terminal residue. The mRNA sequence indicated that Ser is the second N-terminal residue, and the first is Cys. That preparations of the enzyme are mixtures of two polypeptides, one polypeptide being one residue shorter at the N terminus than the other, has been pointed out in the above reference. The signal peptide consists of 43 residues, is rich in basic (4 Lys, 2 Arg) and hydroxylated (4 Thr, 3 Ser) amino acids, and lacks acidic residues.  相似文献   

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The mRNA sequences for two rat pancreatic elastolytic enzymes have been cloned by recombinant DNA technology and their nucleotide sequences determined. Rat elastase I mRNA is 1113 nucleotides in length, plus a poly(A) tail, and encodes a preproelastase of 266 amino acids. The amino acid sequence of the predicted active form of rat elastase I is 84% homologous to porcine elastase 1. Key amino acid residues involved in determining substrate specificity of porcine elastase 1 are retained in the rat enzyme. The activation peptide of the zymogen does not appear related to that of other mammalian pancreatic serine proteases. The mRNA for elastase I is localized in the rough endoplasmic reticulum of acinar cells, as expected for the site of synthesis of an exocrine secretory enzyme. Rat elastase II mRNA is 910 nucleotides in length, plus a poly(A) tail, and encodes a preproenzyme of 271 amino acids. The amino acid sequence is more closely related to porcine elastase 1 (58% sequence identity) than to the other pancreatic serine proteases (33-39% sequence identity). Predictions of substrate preference based upon key amino acid residues that define the substrate binding cleft are consistent with the broad specificity observed for mammalian pancreatic elastase 2. The activation peptide is similar to that of the chymotrypsinogens and retains an N-terminal cysteine available to form a disulfide link to an internal conserved cysteine residue.  相似文献   

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The complete amino acid sequence of a sodium channel from squid Loligo bleekeri has been deduced by cloning and sequence analysis of the complementary DNA. The deduced sequence revealed an organization virtually identical to the vertebrate sodium channel proteins; four homologous domains containing all six membrane-spanning structures are repeated in tandem with connecting linkers of various sizes. A unique feature of the squid Na channel is the 1,522 residue sequence, approximately three fourths of those of the rat sodium channels I, II and III.  相似文献   

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M Misrahi  M Atger  E Milgrom 《Biochemistry》1987,26(13):3975-3982
Complementary DNAs (cDNAs) prepared from messenger RNAs (mRNAs) isolated from endometria of 5 day pregnant rabbits were inserted into the plasmid pBR322. A library of 2400 recombinant plasmid clones was prepared and screened by differential in situ hybridization with cDNAs prepared from mRNAs of rabbits either injected with progesterone or untreated by the hormone. Clones encoding uteroglobin were identified and discarded. Several progesterone-induced and progesterone-repressed clones were identified. One of them corresponded to a relatively frequent mRNA (0.2% of clones in the library) of 2300 nucleotides. The induction of this messenger RNA by progesterone was totally suppressed by the antagonist RU486. This compound displayed a limited agonistic activity when administered alone. A very small increase in mRNA concentration was observed after estradiol administration. The messenger RNA was also found in the liver (where it was constitutively expressed), the ovaries, and the Fallopian tubes of rabbits. A cross-hybridizing messenger RNA was detected in human endometrium during the luteal phase. Sequence analysis showed that the messenger RNA encoded a protein of 370 amino acids with a calculated molecular weight of 40,800. A search in Genbank and National Biomedical Research Foundation data banks showed no identity or marked similarity with previously published DNA or protein sequences.  相似文献   

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The primary structure of rabbit 18S ribosomal RNA was determined by nucleotide sequence analysis of the RNA directly. The rabbit rRNA was specifically cleaved with T1 ribonuclease, as well as with E. coli RNase H using a Pst 1 DNA linker to generate a specific set of overlapping fragments spanning the entire length of the molecule. Both intact and fragmented 18S rRNA were end-labeled with [32P], base-specifically cleaved enzymatically and chemically and nucleotide sequences determined from long polyacrylamide sequencing gels run in formamide. This approach permitted the detection of both cistron heterogeneities and modified bases. Specific nucleotide sequences within E. coli 16S rRNA previously implicated in polyribosome function, tRNA binding, and subunit association are also conserved within the rabbit 18S rRNA. This conservation suggests the likelihood that these regions have similar functions within the eukaryotic 40S subunit.  相似文献   

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