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- Bombesin ; Bombesin receptor ; Chromosomal localization (1)
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- influenza virus ; virion RNA segments ; oligonucleotide mapping ; gene reassortment (1)
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Barley stripe mosaic virus (BSMV) RNA which was previously reported to contain poly(A) sequences (Agranovsky et al., 1978) can be specifically esterified with tyrosine in vitro in the presence of an aminoacyl-tRNA synthetase fraction from wheat embryos. All the three RNA components of the BSMV strain with a three-component genome (Norwich) and both RNA components of a two-component strain (Russian) can be tyrosylated. The poly(A)-containing (bound to oligo(dT)-cellulose) and poly(A)-deficient(not bound to oligo(dT)-cellulose) fractions of BSMV RNA display a similar amino acidaccepting ability. The nucleotide sequence which accepts tyrosine is coupled with the intact genomic polyadenylated BSMV RNA. The viral RNA isolated after sucrose density gradient centrifugation under drastic denaturing conditions retains its aminoacylating activity, which suggests that this activity is not due to the presence in a BSMV RNA preparation of a tyrosine tRNA associated with BSMV RNA. Inhibition of aminoacylation of the 3’-oxidized (treated with sodium metaperiodate) BSMV RNA suggests that the tyrosine-accepting structure is localized at the 3’ terminus of BSMV RNA molecules. It is shown that segments of different lengths obtained upon random fragmentation can be tyrosylated. The 3’-terminal (tyrosine-accepting) poly(A)+ segments can be isolated. The shortest segments of viral RNA capable of being aminoacylated [i.e., containing both tRNA-like structure and poly(A)] consists of approximately 150-200 nucleotides. The analysis of the oligonucleotides derived from individual BSMV RNA components labeled with 32P at the 3’ end revealed two types of 3’-terminal sequences different from poly(A). It is suggested that a poly(A) sequence is intercalated between a 3’-terminal tyrosineaccepting structure and the 5’-terminal portion of poly(A)+ BSMV RNA.
A recombinant plasmid was constructed containing the gene for bovine growth hormone joinea with the regulatory region and the region coding the signal sequence of the Escherichia coli alkaline phosphatase gene. In conditions of phosphorus starvation, which c~s derepression of alkaline phosphatase, expression was shown of the gene for bovine growth hormone, in addition to partial processing and secretion of protein into periplasm.
Structural peculiarities of the S'-end segments of genomic RNA were studied in F potato virus (F-PV) and white clover mosaic virus (WCMV). The methods of affinity chromatography on oligo(dT) cellulose and oligonucleotide mapping revealed a prolonged (up to 210 nucleotides) polyadenyl sequence at the 3'-end of F-PV RNA. A polyadenyl sequence is missing at the 3'end of WCMV RNA. A study of the translation products of WCMV and F-PV RNAs in a oe11-free protein-synthesizing system derived from rabbit reticulocytes showed that polypeptides electrophoretically comigrating with a structural protein of either virus were synthesized alongside high-molecular-weight polypeptides (M\(_r\)\(\approx\) 180-150 kdaltons).
CATIO~IC drugs of different types and structures (antihistaminics, antiarrhythmics, sedatives, opiates, cytostatics and antibiotics, for example) are excreted in mammals by epithelial cells of the renal proximal tubules and by hepatocytes in the liver<sup>1-4</sup>. In the proximal tubules, two functionally disparate transport systems are involved which are localized in the basolateral and luminal plasma membrane and are different from the previously identified neuronal monoamine transporters and A TP-dependent multidrug exporting proteins<sup>1-3,5-12</sup>. Here we report the isolation of a complementary DNA from rat kidney that encodes a 556-amino-acid membrane protein, OCT1, which has the functional characteristics of organic cation uptake over the basolateral membrane of renal proximal tubules and of organic cation uptake into hepatocytes. OCTl is not homologous to any other known protein and is found in kidney, liver and intestine. As OCTl translocates hydrophobic and hydrophilic organic cations of different structures, it is considered to be a new prolotype of polyspecific transporters that are important for drug elimination.
Peptide and polypeptide hormones represent an extensive group of biologically active compounds of important significance for medicine and agriculture. In recent years genetic engineering methods have been used to create strains of microorganisms synthesizing eukaryotic proteins, including hormones and their precursors. The first stage of such developments is the isolation of DNA coding the des~red product. We have accomplished the cloning of the cDNA of a number of polypeptide and peptide hormones of the pituitary of man and domestic animals. The model gene of human calcitonin has also been synthesized and cloned. The obtained genes are being used to develop methods for the microbiological synthesis of human and animal-hormones.
Primary structure is determined of an insertion of a clone isolated from the library of hypophyseal cDNA of cattle by hybridization with a probe specific for prolactin. Analysis of nucleotide sequences showed that in the process of cloning, reorganization occurred in structure of preprolactin cDNA, including an inversion of the 5'-terminal and deletion of the central section of cDNA. Nevertheless, from structure of cDNA, nucleotide sequences can be deduced of extended 5'- and 3'-terminal sections of preprolactin mRNA in cattle with lengths of 257 and 551 nucleotide residues, respectively. When these sequences are compared to those established previously, some differences were found in primary structure. The most important of them is the presence of an additional codon which codes alanine at the position (-22) of the signal peptide. It is suggested that heterogeneity of preprolactin mRNA of cattle in the section coding the signal peptide is the result of alternative splicing, as was shown for preprolactin mRNA in rats.
Results are presented of cloning cDNA of procine growth hormone, analysis of its primary structure, and creation of a construction capable of expression of this cDNA in Esqheriahia coti cells. It is shown that in the population of mRNA coding porcine growth hormone, heterogeneity is noted which is manifested not only at the level of the nucleotide sequence, but also is reflected in the amino acid sequence of the mature hormone.
The homology screening approach has been used to clone a new member of the guanine-nucleotidebinding-protein-coupled receptor superfamily from guinea pig uterus. The cloned cDNA encodes a 399-amino-acid protein and shows the highest amino acid similarity to members of the bombesin receptor family; 52% and 47% similarity to the gastrin-releasing-peptide (GRP) receptor and the neuromedin-B receptor, respectively. Bindingexperiments with the stably transfected LLC-PK<sub>1</sub> cell line expressing the new receptor protein confmned the bombesin-like nature of the cloned receptor. The relative order ofligand affinity, GRP = neuromedin C >> neuromedin B, suggests that the cloned cDNA represents the GRP subtype rather than the neuromedin-B subtype of bombesin receptors. Northern-blot analysis of mRNA species from several guinea-pig tissues showed that the mRNA for the new bombesin receptor subtype is expressed mainly in uteri of pregnant animals.
A cDNA encoding guinea-pig uterine substance P (SP) receptor has been isolated using the homology screening approach. Northern blot analysis reveals that the corresponding mRNA, of approx. 4.8 kb, is expressed in all tissues tested, but predominantly in the uteri of non-pregnant animals; during pregnancy its expression is reduced. The guinea-pig SP receptor was expressed in COS-7 cells and demonstrated relative Iigand affinity in the order: SP >> neurokinin A > neurokinin B.