Physical and functional heterogeneity in TYMV RNA: evidence for the existence of an independent messenger coding for coat protein.

Turnip yellow mosaic virus RNA can be separated into two distinct components of 2 times 10(6) and 300 000 daltons molecular weight after moderate heat treatment in the presence of SDS or EDTA. The two species cannot have arisen by accidental in vitro degradation of a larger RNA, as they both possess capped 5' ends. Analysis of the newly synthesized proteins resulting from translation of each RNA by a wheat germ extract shows that the 300 000 molecular weight RNA can be translated very efficiently into coat protein. When translated in vitro the longer RNA gave a series of high molecular weight polypeptides but only very small amounts of a polypeptide having about the same mass as the coat protein. Thus our results suggest that the small RNA is the functional messenger for coat protein synthesis in infected cells.     

The 21-kDa Polypeptide (VAP21) in the Rabies Virion Is a CD99-Related Host Cell Protein

In our monoclonal antibody (MAb) stocks prepared against the BHK-21 cell antigens, two (#11875 and 28276) recognized a 21-kDa polypeptide (referred to as VAP21) which is efficiently incorporated into the rabies virion. By using these MAbs, we isolated the cDNA clones that encoded a polypeptide of 144 amino acids from our BHK-21 cell cDNA library. Based on the following evidence, the cDNA was assumed to encode a full-length sequence of VAP21 antigen: i) expression of the cDNA in animal cells resulted in the production of a polypeptide recognized by the two MAbs, and its electrophoretic mobility was the same as that of authentic VAP21 antigen; and ii) immunization with the products from the cDNA-transformed E. coli cells raised specific antibodies in rabbits that recognized a 21-kDa polypeptide in the virion. From the deduced amino acid sequence, it is suggested that the VAP21 antigen has a molecular structure of type-I transmembrane protein containing characteristic proline-rich and glycine-rich regions in its ectodomain. Homology searches resulted in finding homologous sequences (totally about 40% homology) in the human MIC2 gene product (CD99; 32-kDa) of T lymphocytes. These results suggest that the VAP21 antigen in the rabies virion is a cellular CD99-related transmembrane protein.     

The use of monoclonal antibodies to quantify the levels of sialoglycoproteins alpha and delta and variant sialoglycoproteins in human erythrocyte membranes.

By using radioiodinated monoclonal antibodies we have estimated that there are about 600 000 copies of sialoglycoprotein alpha (synonym glycophorin A) and 80 000 copies of sialoglycoprotein delta (synonym glycophorin B) per normal human erythrocyte. Erythrocytes expressing the product of only one alpha gene contain about 300 000 copies of alpha/cell. Two erythrocyte types containing alpha-delta hybrid molecules were studied. Those with heterozygous expression of the (alpha-delta)Mi.V gene contain about 100 000 alpha-delta copies per cell, whereas those with heterozygous expression of the En(UK) gene contain about 80 000 alpha-delta copies/cell. Erythrocyte types containing delta-alpha hybrid molecules were also studied. About 200 000 copies of (delta-alpha)Dantu were measured in cells with heterozygous expression of the (delta-alpha)Dantu gene (donor M.P.), whereas about 315 000 copies of the putative (delta-alpha)Dantu hybrid were found on the erythrocytes of donor J.O. [which also have heterozygous expression of the putative (delta-alpha)Dantu gene]. The erythrocytes of donor M.P. have normal levels of alpha, whereas those of donor J.O. have only about half-normal levels. It is proposed that the hybrid sialoglycoprotein of donor J.O. is of alpha-delta-alpha composition [(alpha-delta-alpha)Dantu] rather than delta-alpha and results from a double cross-over analogous to that which gives rise to haemoglobin Parchman.     

Processing Pisum sativum seed storage protein precursors in vitro

The profile of polypeptides separated by SDS-PAGE from seed of major crop species such as pea(Pisum sativum) is complex,resulting from cleavage (processing) of precursors expressed from multiple copies of genes encoding vicilin and legumin,the major storage globulins.Translation in vitro of mRNAs hybridselected from mid-maturation pea seed RNAs by defined vicilin and legumin cDNA clones provided precursor molecules that were cleaved in vitro by a cell-free protease extract obtained from similar stage seed;the derived polypeptides were of comparable sizes to those observed in vivo.The feasibility of transcribing mRNA in vitro from a cDNA clone and cleavage in vitro of the derived translation products was established for a legumin clone,providing a method for determining polypeptide products of an expressed sequence.This approach will also be useful for characterising cleavage site requirements since modifications an readily be introduced at the DNA level.     

Quantitative proteomics of lymphocytes

Lymphocytes are the best-studied higher eukaryote cells. In this report, quantitative relationships of the protein components in resting cell, blast cell and plasma cell types are evaluated. The comparison of these cell types leads to the conclusion that resting cells synthesize about one-twentieth of the protein species as compared to blast cells. Blast cells seem to be metabolically the most robust lymphocyte type. Plasma cells are geared towards synthesis of one main product (antibody in B plasma cells), while most of the synthesis of other protein species (including those for housekeeping and repair) decreases as the messages decay. Although the data presented in this communication allow a meaningful comparison of three cell populations, they are far from providing a full picture. Both silver staining and radiofluorography depict only proteins of high or intermediate abundance. Silver staining misses most proteins present at <10 000 copies/cell, while radiofluorography misses all those proteins with slow turnover (and those with no methionine residue in their sequence). The detection of 1100 spots in the blast cell-related radiofluorograph includes visualization of some 97-99% of protein mass, but some 3900 polypeptide species in the remaining 1-3% of protein mass will pass undetected. This protein mass (0.7-2 pg) reflects some 2500-7500 copies of each of those 3900 polypeptide species that are present in the cell below the detection limit. The work emphasizes that full understanding of cellular function can be achieved only if quantitative aspects of cell inventory are considered.     

Schistosoma mansoni tropomyosin: cDNA characterization, sequence, expression, and gene product localization

We have defined the polypeptide pattern of 3-hr Schistosoma mansoni schistosomula on nonequilibrium two-dimensional gels (NEPHGE). An acidic group of polypeptides with a molecular weight of about 40 kDa and a pI value of around 5.0 (numbered 48/59/53) were identified as antigens on Western blots probed with chronic human infection sera or vaccinated mouse sera. Polypeptides 48/49/53 from silver-stained NEPHGE gels produced antisera that were specific as demonstrated by Western blot analysis and immunoprecipitations of in vitro translation products. A cDNA clone (clone 1) from a S. mansoni adult worm pBR322 library was isolated by using cDNA probes made from size-fractionated mRNA and defined as encoding polypeptide 49 by hybridization selection of the mRNA which was in vitro translated and immunoprecipitated with specific mouse antiserum. A lambda gt 11 expression clone which contained an insert close to the full length mRNA was isolated from a S. mansoni cercariae library. The complete sequence of the mRNA was determined by sequencing the insert of this clone as well as primer extension of total RNA. The only open reading frame coding for 284 amino acids in the 1316 nucleotide sequence showed a 44.76 to 55.44% homology with the amino acid sequences of 18 different tropomyosins from various species. Computer-predicted secondary structure of schistosome tropomyosin was mainly alpha-helix which was very similar to other tropomyosins. Northern analysis showed the mRNA to be about 1.5 kb in size and detectable at much higher levels in the adult worm stage as compared to the cercariae and the egg stages. Western blot analysis likewise showed that greater amounts of tropomyosin were detected in extracts from adult worm stage as compared to extracts from cercariae and egg stages. Immunocytochemical analysis shows that tropomyosin is strongly associated with the tegument of adult worms. The restriction digestion pattern given by genomic Southern analysis suggests the existence of introns and/or multiple gene copies. Thus polypeptide 49, an immunodominant antigen, represents schistosome tropomyosin.     

Isolation and Characterization of a cDNA Encoded an Embryogenic Cell Protein 63 Related to Embryogenesis from Carrot

cDNA library with about 6.0 x 108 plaques per μg phage from carrot ( Daucus carota L. cv. Hammaki) torpedo-shaped embryos was constructed by a eDNA cloning system (λgtl0). A fulllength cDNA for embryogenic cell protein 63 (ECP63), an embryogenic cell protein from carrot with a relative molecular weight of 63 000, was isolated from a eDNA library using PCR-amplified DNA as a probe. The nucleotide sequence of ECP63 cDNA was 1 989 bp in length. The cDNA encoded a polypeptide of 569 amino acids, and the calculated molecular weight of this pelypeptide was 62 000. The Northern blot analysis using labeled full-length ECP63 cDNA as a probe showed that the gene for ECP63 was expressed in embryogenic cells, globular, heart-, and torpedo-shaped embryos, but not in seedlings and non-embryogenic cells.     

Evolutionary analyses of non‐family genes in plants

There are a large number of ‘non‐family’ (NF) genes that do not cluster into families with three or more members per genome. While gene families have been extensively studied, a systematic analysis of NF genes has not been reported. We performed comparative studies on NF genes in 14 plant species. Based on the clustering of protein sequences, we identified ~94 000 NF genes across these species that were divided into five evolutionary groups: Viridiplantae wide, angiosperm specific, monocot specific, dicot specific, and those that were species specific. Our analysis revealed that the NF genes resulted largely from less frequent gene duplications and/or a higher rate of gene loss after segmental duplication relative to genes in both low‐copy‐number families (LF; 3–10 copies per genome) and high‐copy‐number families (HF; >10 copies). Furthermore, we identified functions enriched in the NF gene set as compared with the HF genes. We found that NF genes were involved in essential biological processes shared by all plant lineages (e.g. photosynthesis and translation), as well as gene regulation and stress responses associated with phylogenetic diversification. In particular, our analysis of an Arabidopsis protein–protein interaction network revealed that hub proteins with the top 10% most connections were over‐represented in the NF set relative to the HF set. This research highlights the roles that NF genes may play in evolutionary and functional genomics research.     

Isolation and characterization of rat cDNA clones for two distinct thyroid hormone receptors

Two distinct v-erbA-related cDNA clones representing the products of different genes were isolated from a rat liver cDNA library. The first, rc-erbA-alpha, was 82% identical to v-erbA and encoded a polypeptide with a calculated molecular mass of 45,000 daltons. This cDNA clone arises from the same gene product as a v-erbA-related cDNA isolated from rat brain by Thompson et al. (Thompson, C. C., Weinberger, C., Lebo, R., and Evans, R. (1987) Science 237, 1610-1614). The second cDNA clone, rc-erbA-beta, was 76% identical to v-erbA and encoded a polypeptide with a calculated molecular mass of 52,000 daltons. Both rc-erbA-alpha and rc-erbA-beta translational products bound 3,5,3'-triiodo-L-thyronine with affinities equal to each other (Kd approximately equal to 0.4 nM) and comparable to the nuclear thyroid hormone receptor extracted from rat liver. The relative affinities of a series of thyroid hormone analogs for both translational products were also identical. In various tissues and cell lines, the relative levels of rc-erbA-beta RNA, but not rc-erbA-alpha RNA, correlated with measurements of nuclear 3,5,3'-triiodo-L-thyronine binding sites. Based on this correlation, we suggest that rc-erbA-beta may encode the "classical" nuclear thyroid hormone receptor, whereas rc-erbA-alpha may encode an isoreceptor species with differing functional properties.     

Storage protein precursor polypeptides in cotyledons of Pisum sativum L. Identification of, and isolation of a cDNA clone for, an 80000-Mr legumin-related polypeptide

An 80 000-Mr polypeptide, which bound to anti-legumin IgG, was detected among labelled polypeptides from cotyledons at late stages of development. When poly(A)-containing RNA from similar cotyledons was translated in a cell-free protein-synthesizing system, an 80 000-Mr polypeptide was also detected. Immunoprecipitation of translation products with anti-legumin IgG showed that, in addition to the major legumin precursor polypeptides of Mr approximately 60 000, the 80 000-Mr polypeptide was specifically immunoprecipitated. A cDNA clone, pCD32, was found to select an RNA coding for an 80 000-Mr polypeptide in hybrid-selection experiments. Additional minor polypeptides of Mr 63 000 and 65 000 were present in translation products of RNA selected by pCD32; all three polypeptides were immunoprecipitated by anti-legumin IgG. Thermal elution of RNAs bound to pCD32 showed that the affinity of pCD32 to the RNA coding for the 80 000-Mr polypeptide was greater than to the RNAs coding for the 63 000-Mr and 65 000-Mr polypeptides. In similar hybrid-selection experiments, another cDNA clone, pCD40, selected RNAs coding predominantly for polypeptides of Mr 63 000 and 65 000. A minor polypeptide of Mr 80 000 was also detected among these products; again all three polypeptides were immunoprecipitated by anti-legumin IgG. Peptide mapping revealed close similarities between the 80000-Mr polypeptide and the 63 000-Mr/65 000-Mr polypeptides obtained by translation of RNAs selected by pCD32. There were similarities also between maps obtained from translation products of RNA selected by pCD32 and those obtained from anti-legumin IgG immunoprecipitates of total translation products of poly(A)-containing RNA.     

The subunit characterization of Callinectes sapidus hemocyanin.

Hemocyanin from the blue crab, Callinectes sapidus, sediments at 25.7 S and has a native molecular weight of 940 000 +/- 20 000. Under solution conditions of increased pH (approximately 10) or ionic strength, the native molecule dissociates to a 17 S species. Reversal of this dissociation was unsuccessful. At pH 10 and with the removal of Mg2+, the 17 S species reversibly dissociates to form a subunit species which sediments at 6 S. A comparison of the circular dichroic spectra of the 25.7 S and 6 S hemocyanins suggests that little happens to the structural integrity of the polypeptide backbone upon the two dissociations. Molecular weight estimations under reducing and denaturing conditions indicate that the 6 S hemocyanin species represents the constituent polypeptide chain of the protein molecule. Chemical analysis suggests the presence of a small amount, less than 3%, of carbohydrate bound to the polypeptide chain. Electrophoresis of the hemocyanin in the presence of sodium dodecyl sulfate or urea reveals two major electrophoretic species of either slightly different chemical composition or slightly different polypeptide chain length.     

Majority of random cDNA clones correspond to single loci in the tomato genome

Summary The number of loci corresponding to each of 34 unique, random cDNA clones has been determined for the diploid plant species Lycopersicon esculentum (tomato) (2n=24). Fifty-three percent of the clones are homologous to loci represented only once in the tomato chromosomes. Thirty-two percent of the clones correspond to two genetically independent loci. The remaining clones belong to gene families represented by 3–5 loci. To determine the number of gene copies per locus, reconstruction experiments were performed on six of the single locus clones. The majority of these loci were estimated to contain only 1 or 2 copies of the gene. These results support the concept that the majority of structural genes in this diploid plant species are arranged in single loci and present in low copy number. Multigene families, such as those for the small subunit of ribulose bisphosphate carboxylase, chlorophyll a/b binding polypeptide and actin are exceptions to this rule.     

Mitochondrial pseudogenes are pervasive and often insidious in the snapping shrimp genus Alpheus

Here we show that multiple DNA sequences, similar to the mitochondrial cytochrome oxidase I (COI) gene, occur within single individuals in at least 10 species of the snapping shrimp genus Alpheus. Cloning of amplified products revealed the presence of copies that differed in length and (more frequently) in base substitutions. Although multiple copies were amplified in individual shrimp from total genomic DNA (gDNA), only one sequence was amplified from cDNA. These results are best explained by the presence of nonfunctional duplications of a portion of the mtDNA, probably located in the nuclear genome, since transfer into the nuclear gene would render the COI gene nonfunctional due to differences in the nuclear and mitochondrial genetic codes. Analysis of codon variation suggests that there have been 21 independent transfer events in the 10 species examined. Within a single animal, differences between the sequences of these pseudogenes ranged from 0.2% to 20.6%, and those between the real mtDNA and pseudogene sequences ranged from 0.2% to 18.8% (uncorrected). The large number of integration events and the large range of divergences between pseudogenes and mtDNA sequences suggest that genetic material has been repeatedly transferred from the mtDNA to the nuclear genome of snapping shrimp. Unrecognized pseudogenes in phylogenetic or population studies may result in spurious results, although previous estimates of rates of molecular evolution based on Alpheus sister taxa separated by the Isthmus of Panama appear to remain valid. Especially worrisome for researchers are those pseudogenes that are not obviously recognizable as such. An effective solution may be to amplify transcribed copies of protein-coding mitochondrial genes from cDNA rather than using genomic DNA.     

Monitoring of Gene Expression Profiles and Isolation of Candidate Genes Involved in Pollination and Fertilization in Rice (Oryza Sativa L.) with a 10K cDNA Microarray

To monitor gene expression profiles during pollination and fertilization in rice at a genome scale, we generated 73,424 high-quality expressed sequence tags (ESTs) derived from the green/etiolated shoot and pistil (0-5 h after pollination, 5hP) of rice, which were subsequently used to construct a cDNA microarray containing ca. 10 000 unique rice genes. This microarray was used to analyze gene expression in pistil unpollinated (UP), 5hP and 5DAP(5 days after pollination), anther, shoot, root, 10-day-old embryo (10EM) and 10-day-old endosperm (10EN). Clustering analysis revealed that the anther has a gene-expression profile more similar to root than to pistil and most pistil-preferentially expressed genes respond to pollination and/or fertilization. There are 253 ESTs exhibiting differential expression (e +/- 2-fold changes) during pollination and fertilization, and about 70% of them can be assigned a putative function. We also recovered 20 genes similar to pollination-related and/or fertility-related genes previously identified as well as genes that were not implicated previously. Microarray and real-time PCR analyses showed that the array sensitivity was estimated at 1-5 copies of mRNA per cell, and the differentially expressed genes showed a high correlation between the two methods. Our results indicated that this cDNA microarray constructed here is reliable and can be used for monitoring gene expression profiles in rice. In addition, the genes that differentially expressed during pollination represent candidate genes for dissecting molecular mechanism of this important biological process in rice.     

The properties of the large subunit of maize ribulose bisphosphate carboxylase/oxygenase synthesised in Escherichia coli

The maize chloroplast gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase has been expressed in Escherichia coli in vivo. This enables the properties of the native large-subunit polypeptide to be examined in the absence of small-subunit polypeptides, and avoids the use of denaturing agents. The product synthesised in bacteria is slightly larger (Mr 54300) than the form present in the chloroplast (Mr 53 300), suggesting the involvement of a precursor polypeptide. In addition several smaller polypeptides are synthesised, predominantly of molecular mass 41 and 30 kDa, but also some of 44 and 12-14 kDa. Pulse-chase experiments with [35S]methionine indicate that all the immunoprecipitable polypeptides are stable. The smaller products are probably the result of premature termination of translation. Virtually all of the large subunits are insoluble, whether synthesised at levels of 100-200 molecules per cell, or up to 60 000 molecules per cell. A small amount of the full-length polypeptide is soluble, but the major soluble product, as determined by sucrose gradient centrifugation, is a polypeptide of molecular mass 12-14 kDa. Ribulose bisphosphate carboxylase activity was undetectable in cell extracts, and binding of a mixture of the radiolabelled transition state analogues carboxyribitol 1,5-bisphosphate and carboxyarabinitol 1,5-bisphosphate could not be detected. It is proposed that other components are required to prevent the large subunit from adopting an inactive, insoluble conformation after, or during, synthesis.     

Cytosolic triosephosphate isomerase is a single gene in rice.

A cDNA clone encoding rice (Oryza sativa L.) cytosolic triosephosphate isomerase (TPI), an important glycolytic enzyme, was isolated and characterized. The clone (pRTPI-6) contains an open reading frame of 759 base pairs, encoding a polypeptide chain of 253 amino acid residues (M(r) 27,060). The identity of this clone was defined by its high homology (85% nucleotide sequence and 89% amino acid sequence identical match) with a maize mRNA sequence encoding the cytosolic TPI and with TPIs from other species. Genomic DNA blot analysis using the cDNA as a probe showed that the cytosolic TPI gene is present as a single copy per haploid rice genome, as opposed to that found in maize, in which multiple TPI gene copies exist. A single TPI mRNA species of about 1100 nucleotides was detected by gel blot hybridization analysis of RNA isolated from root, culm, and leaf tissues, indicating that its expression is ubiquitous. Based on sequence comparison and molecular analysis, we propose that the chloroplast-located TPI may be encoded by divergent structural nuclear genes in rice.