N‐Terminal Sequence and Main Characteristics of Atlantic Salmon (Salmo salar) Albumin

Abstract

Atlantic salmon (Salmo salar) serum albumin was purified from plasma and its N‐terminal sequence determined. Atlantic salmon albumin is the predominant plasma protein, negatively charged, at pH 8.6. Albumin was purified to >95% purity which yielded a single band on SDS‐PAGE and agarose gel electrophoresis. The molecular weight of the purified albumin was approximately 6,5 kDa. The N‐terminal sequence of Atlantic chinook salmon albumin was consistent with that predicted from its previously determined cDNA sequence and was identical to that of salmon (Oncorhynchus tshawytscha) albumin through the first 15 residues. However, the fact that the actual N‐terminus was different from that predicted from cDNA sequence indicates that Atlantic salmon albumin, like chinook salmon albumin, lacks a propeptide.     

Molecular cloning of cDNA sequences transcribed from mouse liver endoplasmic reticulum poly(A)mRNA

Poly(A)mRNA was isolated from the endoplasmic reticulum (ER) of male BALB/c mice and used as the template for cDNA synthesis. Double-stranded (ds) cDNA was cloned in a bacterial plasmid and 21 clones were selected for further study. Six clones have been identified by hybrid selection of mRNA, translation of the mRNA in a template-dependent system, polyacrylamide gel electrophoresis and specific immunoprecipitation. Four code for the major urinary protein of the mouse (MUP), one for serum albumin and one for alpha 1-antitrypsin. A seventh codes for an unidentified polypeptide with Mr = 27000. Initial-rate hybridisation kinetics of immobilised cloned sequences with end-labelled mRNA showed that serum albumin mRNA is most abundant in the population of mRNA species present in the endoplasmic reticulum fraction of female BALB/c liver. MUP mRNA is also very abundant. The alpha 1-antitrypsin mRNA and the mRNA specifying the 27000-dalton protein are about 5 times less abundant than serum albumin mRNA on a molar basis.     

Changes in expression of albumin and alpha-fetoprotein genes during rat liver development and neoplasia.

Albumin mRNA was isolated and purified from rat liver polysomes by a combination of immunoprecipitation of specific polysomes, poly(U)-Sepharose 4B chromatography, and fractionation of the resulting poly(A)-containing RNA on a sucrose gradient. alpha-Fetoprotein (AFP) mRNA was isolated from Morris hepatoma 7777 by a similar procedure. The purity of the mRNA preparations was determined by analytical gel electrophoresis under denaturing conditions, analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polypeptides synthesized in a wheat germ cell-free system, and the kinetics of hybridization to cDNA transcribed from albumin mRNA and AFP mRNA. The albumin mRNA possessed a chain length of approximately 2265 nucleotides and the AFP mRNA possesed a length of approximately 2235 nucleotides when examined under stringent denaturing conditions on agarose gels containing 10 mM methylmercury hydroxide. Analysis of poly(A) content by a hybridization assay with [3H]poly(U) revealed the presence in albumin mRNA of a poly(A) region containing approximately 100 adenosine residues. The AFP mRNA preparation was found to contain an average poly(A) tract of approximately 190 bases. Thus, albumin mRNA appears to contain approximately 330 untranslated nucleotides, and AFP mRNA appears to contain a similar number (approximately 285) of noncoding, nonpoly(A) bases. The purified albumin and AFP mRNA's were used as templates for synthesis of full-length cDNA hybridization probes. Both of the probes selectively hybridized to their templates with kinetics expected for single RNA species the sizes of albumin and AFP mRNA. ROt analysis was used to quantitate albumin and AFP mRNA sequences during normal liver postnatal development and liver oncogenesis. The number of polysomal AFP mRNA molecules per liver was found to drastically decrease during the first weeks of postnatal life, concomitant with a decline in the AFP synthetic capacity of the livers and in the serum concentrations of AFP. During this period, the concentration of albumin mRNA molecules per cell in the liver remained at high, approximately constant levels. In Morris hepatoma 7777, the concentration of AFP-specifying sequences was at least 10(3)-fold higher than that found in normal adult liver, whereas the content of albumin nRNA was four- to five-fold lower. These changes in concentration of albumin and AFP mRNA sequences closely correlated with a parallel variation in the specific protein synthetic capacity of the tissues.     

Molecular mechanisms for changes in hepatic protein synthesis induced by schistosomiasis infection in mice

Mice infected with Schistosoma mansoni and littermate controls were evaluated serially for 12 weeks. Infected mice gained weight at the same rate as controls, but starting with the sixth week their livers became enlarged with granulomas and fibrous tissue, and they developed hypoalbuminemia. To evaluate the regulation of the albumin and type I collagen gene expression, total RNA was isolated from infected and control mice and translated in an mRNA-dependent rabbit reticulocyte lysate system. Protein synthesis was decreased 1.5-3-fold with RNA from infected vs. control liver. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell-free products showed a reduction in albumin but an increase in type I procollagen synthesis in infected mice. Immunoprecipitation of the cell-free product confirmed that albumin synthesis was reduced in greater proportion than other liver proteins in schistosome-infected mice. Hybridization of RNA from infected liver with cloned mouse albumin cDNA (pmalb-2) demonstrated a reduction in albumin mRNA to 37% of control, while hybridization with a chick type I pro alpha 2 collagen cDNA probe (pCg-45) revealed increased procollagen mRNA in infected liver beginning at 6 weeks postinfection. These results suggest that in murine schistosomiasis a reduction in biologically active albumin mRNA results in decreased albumin synthesis and may be responsible in part for hypoalbuminemia. In addition, increased collagen mRNA is associated with increased collagen synthesis during hepatic fibrosis.     

cDNA cloning of mRNAs induced in resistant barley during infection by Erysiphe graminis f.sp. Hordei

Summary Near-isogenic cultivars of Hordeum vulgare which differ for the Mlp gene for resistance to Erysiphe graminis f.sp. hordei were inoculated with race 3 of this pathogen and in vitro translation products of mRNA populations compared by 2-dimensional gel electrophoresis and fluorography. This revealed the presence of new mRNA species in infected leaves compared to non-inoculated controls. These new mRNA species were more abundant in resistant leaves than susceptible leaves. A cDNA library was prepared from poly(A)⁺RNA isolated from infected leaves carrying the Mlp gene for resistance (cvMlp). The library was screened by differential hybridization using [³²P]-labelled cDNA prepared from poly(A)⁺RNA of both control and infected leaves. Six cDNA clones showing greater hybridization to cDNA prepared from infected leaves were selected. These six cDNA clones hybridized to DNA isolated from barley leaves but not to DNA from conidia of the fungus. In Northern blot analysis of RNA from infected leaves the six cDNA clones each hybridized to mRNA species of different size. Translation products for three of the cDNA clones corresponded to infection-related translation products identified on 2-dimensional fluorograms. The cDNA clones were used to study the kinetics of host mRNA induction during infection of the near-isogenic cultivars of barley. The host mRNA species corresponding to the cDNA clones were induced prior to 24 h after inoculation during the primary penetration processes. In addition the mRNAs corresponding to four of the cDNA clones increased to greater amounts in cvMlp than in the near-isogenic susceptible cultivar (cvmlp) over a 2-d period following inoculation. These results suggest that the Mlp gene has a regulatory role in host gene expression resulting in enhanced expression of several host mRNA species following infection by the powdery mildew fungus.     

北京鸭珠蛋白mRNA的分离纯化及其cDNA的合成

 从人工贫血的北京鸭网织红细胞中直接提取总RNA,经Oligo(dT)-纤维素柱层析分离获得珠蛋白mRNA,并经蔗糖密度梯度离心首次得到了电泳单一条带的北京鸭球蛋白mRNA。从凝胶电泳以及蔗糖密度梯度离心鉴定其沉降系数为9S。在麦胚无细胞体外翻译体系中测定了它们的蛋白翻译活力。鸭珠蛋白mRNA促进了~3H-亮氨酸参入新生蛋白的活力,达到对照组的10倍。所翻译的蛋白产物在SDS-聚丙烯酰胺凝胶上的电泳行为与天然鸭珠蛋白一致。 经Oligo(dT)-纤维素及蔗糖密度梯度离心提纯的珠蛋白mRNA,在AMV反转录酶及DNA聚合酶的作用下,分别合成了单链及双链cDNA。其双链链长,经凝胶电泳分析,约为500碱基对。     

N-Terminal Sequence and Main Characteristics of Atlantic Salmon (Salmo salar) Albumin

Atlantic salmon (Salmo salar) serum albumin was purified from plasma and its N-terminal sequence determined. Atlantic salmon albumin is the predominant plasma protein, negatively charged, at pH 8.6. Albumin was purified to >95% purity which yielded a single band on SDS-PAGE and agarose gel electrophoresis. The molecular weight of the purified albumin was approximately 6,5 kDa. The N-terminal sequence of Atlantic chinook salmon albumin was consistent with that predicted from its previously determined cDNA sequence and was identical to that of salmon (Oncorhynchus tshawytscha) albumin through the first 15 residues. However, the fact that the actual N-terminus was different from that predicted from cDNA sequence indicates that Atlantic salmon albumin, like chinook salmon albumin, lacks a propeptide.     

Biochemical changes in progressive muscular dystrophy. XIV. Skeletal muscle myosin mRNA translatability in dystrophic mice

Variations in the content and translatability of the poly(A)+ RNA and mRNA molecules coding for myosin (M) were studied in the hind leg muscles of genetically dystrophic mice. The poly(A)+ RNA content of total skeletal muscle failed to increase normally during progression of the disease. M mRNA, isolated from dystrophic normally during progression of the disease. M mRNA, isolated from dystrophic murine muscle poly(A)+ RNA, was mostly found to be associated with the 26S RNA species. The translation of M mRNA in an in vitro heterologous wheat germ system was lower at 8 and 16 weeks in the dystrophic group as compared with the controls. Analysis of the translation products via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and densitometric autoradiographic tracing demonstrated the gradual disappearance of a protein band corresponding to M, the major component of skeletal muscle. cDNA was synthesized, using M mRNA that was isolated and purified from normal and dystrophic mouse muscle as a template. Total radioactivity was measured in some cDNA fractions produced from normal and dystrophic mouse muscle, while other fractions were utilized for separation and sizing of cDNA by disc gel electrophoresis. The cDNA from normal muscle was hybridized with M mRNA from normal and 16-week-old dystrophic mouse muscles. The cDNA probe, hybridization experiments, and studies involving the content and synthesis of M mRNA suggest that murine muscular dystrophy elicited a shorter species of mRNA or shorter sequences of the same species of mRNA coding for M. Not all poly(A)+ mRNA sequences coding for M, found in control mice, were present in their dystrophic counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and functional analysis of a cDNA encoding a starch synthase from potato (Solanum tuberosum L.) that is predominantly expressed in leaf tissue

Three isoforms of starch synthase (SS) were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. A cDNA encoding SSI from rice was used as a probe to clone a corresponding cDNA from potato. The deduced amino acid sequence identified the protein as an SS from potato with an M_r of 70.6 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SSI. An analysis of the expression pattern of the gene indicated that SSI is predominantly expressed in sink and source leaves, and, to a lower extent in tubers. In several independent transgenic potato lines, where the expression of SSI was repressed using the antisense approach, the activity of a specific SS isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction in the amount of this isoform of SS did not lead to any detectable changes in starch structure, probably due to the fact that this isoform only represents a minor activity in potato tubers. Received: 19 August 1998 / Accepted: 17 December 1998     

Albumin is encoded by 2 messenger RNAs in Xenopus laevis

A cDNA clone library was prepared from liver poly(A) RNA pf non-estrogenized Xenopus laevis. Albumin coding sequences were screened by hybridization to a cDNA prepared from poly(A) RNA enriched by sucrose density gradient centrifugation, and by a sensitive solid-phase radioimmunoassay to detect clones that contain templates for albumin antigenic determinants. Nine clones were obtained by this approach, and all but one have the cDNA inserted in phase with the beta-lactamase gene of pBR322. Mapping of these clones with restriction endonucleases yielded 2 distinct patterns, suggestive of heterogeneity in the coding sequences. This was confirmed by heteroduplex analyses of hybrids formed between clones representative of each of the 2 classes. Both classes of albumin cDNA clones were used to select mRNAs of the same size (2.3kb) that code for peptides that are indistinguishable by SDS gel electrophoresis. Examination of the organization of the albumin genes by blot hybridization of the cDNA clones to restriction fragments of Xenopus DNA failed to detect any differences at the genomic level. The considerable diversity of the albumin cDNAs is suggestive of a multiplicity of albumin genes, rather than differential processing of a common precursor RNA.     

A reliable method for extraction of RNA from various conifer tissues

Summary A simple and efficient procedure suitable for extraction of high-quality RNA from cultured conifer tissues, somatic embryos, zygotic embryos, needles, stem and root tissues was developed. It produced from 100 g up to 700 g total RNA per gram tissue dependent on the types of tissues used. RNA quality was estimated by spectrophotometry, agarose gel electrophoresis, in vitro translation of mRNA, cDNA synthesis and Northern blot analysis. The method also worked well with Arabidopsis thaliana and tobacco tissues.Abbreviations CTAB cetyltrimethylammonium bromide - DEPC diethylpyrocarbonate - PVP polyvinylpyrrolidone - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

The nucleotide sequence of a complete chicken delta-crystallin cDNA

The nucleotide sequence of a full length cDNA of delta-crystallin mRNA from chicken lens has been determined using a delta-crystallin cDNA clone (pB delta 11), which represents the mRNA sequence of 1530 nucleotides from the poly(A) junction but does not contain the 5'-terminal sequence of 44 nucleotides of the mRNA. The 5'-terminal sequence of the mRNA, absent in the cDNA clone, has been determined with a stretch of cDNA sequence by the primer extension procedure. The amino acid sequence deduced from the nucleotide sequence is consistent with the amino acid sequences of several tryptic peptides, the total amino acid composition, and the mol. wt. of delta-crystallin estimated by SDS-polyacrylamide gel electrophoresis. The computer-assisted analysis predicts high alpha-helical content throughout the polypeptide. Sequence analyses have revealed that gene 1 encodes the mRNA from which the cDNA clone was derived.     

Isolation of globin messenger RNA of Xenopus laevis

The polypeptide chains of Xenopus laevis hemoglobin have been analyzed by sodium dodecyl sulfate (SDS) and acid-urea gel electrophoresis. Four components can be distinguished, each having an approximate molecular weight of 13,000 daltons. Messenger RNA coding for the globin chains has been isolated and characterized. In a denaturing acrylamide gel the mRNA has an approximate molecular weight of 250,000 daltons. The complexity of the RNA is consistent with the presence of four different mRNA molecules, each of this molecular weight. When the mRNA is assayed in a wheat germ in vitro translation system, four polypeptides are synthesized corresponding to the four globin subunits. The relative proportion of the four synthesized polypeptides appears to vary according to the developmental stage of the red blood cells used for mRNA isolation. Hybridization of a complementary DNA (cDNA) copy of the globin mRNA to Xenopus laevis DNA in DNA excess indicates that each of the globin genes is present in one to three copies per haploid genome.     

Identification of a glucose-6-phosphate isomerase involved in adaptation to salt stress of <Emphasis Type="Italic">Dunaliella salina</Emphasis>

The unicellular green alga Dunaliella salina is a recognized model for studying plant adaptation to high salinity. To isolate some salt-induced proteins at proteomics levels and to identify their expressions at gene levels, algal cells at logarithmic phase cultured in 1.5 and 3.5 M NaCl media were harvested for protein extraction. Solubilized proteins were applied to two-dimensional gel electrophoresis (2-DE) and analyzed by ImageMaster 2D Platinum software. Twenty-one protein spots whose intensities were elevated threefold to 13-fold at 3.5 M NaCl as compared to 1.5 M NaCl were analyzed by matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry. One salt-induced protein isolated from the 2-DE gels was identified as a glucose-6-phosphate isomerase (GPI) from D. salina (DsGPI). A full-length cDNA of DsGPI was obtained using rapid amplification of cDNA end technique, and it was shown by heterologous expression to encode a protein with a molecular weight consistent with the protein spot in the 2-DE gels. Real-time quantitative RT-PCR demonstrated that the mRNA of DsGPI was induced up to eightfold (P < 0.01) by 2.5 M and 14-fold higher (P < 0.01) by 3.5 M NaCl than by 1.5 M NaCl, respectively. It is concluded that the protein isolated through 2-DE is indeed DsGPI and that the DsGPI gene may be involved in adaptation to high salinity.     

Restriction landmark cDNA scanning (RLCS): a novel cDNA display system using two-dimensional gel electrophoresis.

We have developed a new method, designated restriction landmark cDNA scanning (RLCS), which displays many cDNA species quantitatively and simultaneously as two-dimensional gel spots. In this method cDNA species of uniform length were prepared for each mRNA species using restriction enzymes. After the restriction enzyme sites were radiolabeled as landmarks, the labeled fragments were subjected to high resolution two-dimensional gel electrophoresis. In analyses of cDNA samples from adult mouse liver and brain (cerebral cortex, cerebellum and brain stem) we detected approximately 500 and >1000 discrete gel spots respectively of various intensities at a time. The spot patterns of the three brain regions were very similar, although not identical, but were quite different from the pattern for the liver. RNA blot hybridization analysis using several cloned spot DNAs as probes showed that differences in intensity of the spots among RLCS profiles correlated well with expression levels of the corresponding mRNA species in the brain regions. Because the spots and their intensities reflect distinct mRNA species and their expression level respectively, the RLCS is a novel cDNA display system which provides a great deal of information and should be useful for systematic documentation of differentially expressed genes.