Two divergent cellular src genes are expressed in Xenopus laevis.

Genomic and cDNA clones of the X. laevis src gene have been isolated and characterized by hybridization and DNA sequence analyses. The haploid genome of X. laevis contains two src genes, which can be distinguished from one another by virtue of sequence divergence in the 3' untranslated regions. Both of the genes are functional as indicated by the fact that oocytes contain RNAs transcribed from each of the genes. The two genes each encode an RNA which is 3.3 kb in length, or twice the length required to encode the 60,000 dalton src protein (pp60). Sequence analysis of the cDNA clones revealed that nearly all of the non-coding sequence is located at the 3' end. The availability of sequence data from cDNA clones has also made it possible for the first time to identify with certainty the carboxyl terminal sequence of a cellular pp60 molecule.     

Structural analysis of the three vitellogenin genes in Drosophila melanogaster.

Genomic fragments coding for sequences expressed as abundant mRNA in female Drosophila melanogaster were isolated from a lambda library. Hybridization of these clones to polytene chromosomes. in situ, identified four which mapped to X chromosomal region 9A to 9B, the locus for yolk proteins 1 and 2 (Ypl,2) and two which mapped to 12A6-7 to 12D3, the locus for Yp3. These clones were mapped with restriction enzymes, and the coding regions and regions of homology determined by Southern blots probed with cDNA, 5'-end-labelled RNA and nick-translated DNA. Heteroduplex and R-loop mapping confirmed that three of the clones carried two genes separated by about 1.4 kb and oriented in opposite directions. Southern blots probed with cDNA made from alkali-hydrolyzed RNA showed that these genes had their 5' ends next to each other. All 3 genes show homology to each other and have a main coding region of about 1.3 kb, the approximate size for the mRNAs.     

Three distinct ribonucleoproteins from tobacco chloroplasts: each contains a unique amino terminal acidic domain and two ribonucleoprotein consensus motifs.

Chloroplasts contain their own genetic system. Eighteen different split genes have been found among approximately 130 chloroplast genes from higher plants. However, little is known about the chloroplast splicing system. Mammalian heterogeneous nuclear ribonucleoproteins (hnRNP proteins) have been shown to be involved in splicing. We applied a purification procedure developed for HeLa cell hnRNP proteins, which uses a single-stranded DNA (ssDNA) affinity column, directly to the tobacco chloroplast lysate to isolate their chloroplast counterparts. Four proteins (mol. wt approximately 30 kd) bound strongly to the column. The amino-terminal sequences of three of them were determined and their cDNA clones were isolated from a tobacco leaf cDNA library. Sequence analysis of these clones revealed that all three proteins contain two ribonucleoprotein consensus sequences (RNP-CS), confirming their ribonucleoprotein (RNP) nature. The presence of putative transit peptides in their predicted protein sequences, and an in vitro import experiment confirmed they are located in the chloroplast. This is the first report of organellar proteins containing RNP-CS. In addition, these three chloroplast proteins have a very acidic amino-terminal domain, a novel feature among RNP proteins identified so far. They are expressed both in leaves and roots; their mRNA levels showed different light modulation in mature leaves. The three proteins might be involved in splicing and/or processing of chloroplast RNAs.     

Characterization of long cDNA clones from human adult spleen. II. The complete sequences of 81 cDNA clones.

To accumulate information on the coding sequences (CDSs) of unidentified genes, we have conducted a sequencing project of human long cDNA clones. Both the end sequences of approximately 10,000 cDNA clones from two size-fractionated human spleen cDNA libraries (average sizes of 4.5 kb and 5.6 kb) were determined by single-pass sequencing to select cDNAs with unidentified sequences. We herein present the entire sequences of 81 cDNA clones, most of which were selected by two approaches based on their protein-coding potentialities in silico: Fifty-eight cDNA clones were selected as those having protein-coding potentialities at the 5'-end of single-pass sequences by applying the GeneMark analysis; and 20 cDNA clones were selected as those expected to encode proteins larger than 100 amino acid residues by analysis of the human genome sequences flanked by both the end sequences of cDNAs using the GENSCAN gene prediction program. In addition to these newly identified cDNAs, three cDNA clones were isolated by colony hybridization experiments using probes corresponding to known gene sequences since these cDNAs are likely to contain considerable amounts of new information regarding the genes already annotated. The sequence data indicated that the average sizes of the inserts and corresponding CDSs of cDNA clones analyzed here were 5.0 kb and 2.0 kb (670 amino acid residues), respectively. From the results of homology and motif searches against the public databases, functional categories of the 29 predicted gene products could be assigned; 86% of these predicted gene products (25 gene products) were classified into proteins relating to cell signaling/communication, nucleic acid management, and cell structure/motility.     

Nad+-dependent isocitrate dehydrogenase from Arabidopsis thaliana. Characterization of two closely related subunits

Two cDNA clones which appear to encode different subunits of NAD⁺-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.41) were identified by homology searches from the Arabidopsis EST database. These cDNA clones were obtained and sequenced; both encoded full-length messages and displayed 82.7% nucleotide sequence identity over the coding region. The deduced amino acid sequences revealed preprotein lengths of 367 residues, with an amino acid identity of 86.1%. Genomic Southern blot analysis showed distinct single-copy genes for both IDH subunits. Both IDH subunits were expressed as recombinant proteins in Escherichia coli, and polyclonal antibodies were raised to each subunit. The Arabidopsis cDNA clones were expressed in Saccharomyces cerevisiae mutants which were deficient in either one or both of the yeast NAD⁺-dependent IDH subunits. The Arabidopsis cDNA clones failed to complement the yeast mutations; although both IDH-I and IDH-II were expressed at detectable levels, neither protein was imported into the mitochondria.     

The pattern of expression of the Xenopus laevis tadpole alpha-globin genes and the amino acid sequence of the three major tadpole alpha-globin polypeptides

We have isolated cDNA clones derived from three tadpole alpha-globin mRNAs of Xenopus laevis. The entire nucleotide sequence of the three mRNAs has been determined from the cDNA clones and is presented together with the deduced amino acid sequence of the encoded polypeptides. Two of the three polypeptide sequences are 96% homologous whilst the third sequence is highly diverged, with only a 72% homology. The three tadpole alpha-globin genes are all similarly diverged from the two X. laevis adult alpha-globin genes with which they display approximately 50% homology. Analysis of several independent clones from each class of tadpole alpha-globin sequence reveals a very high degree of coding region polymorphism for each of the three corresponding genes. Using the cloned DNA sequences as hybridisation probes, we have analysed the expression of the corresponding genes during larval development. We show that all three genes are activated simultaneously early in development and that thereafter all three are expressed at an approximately equivalent level. A fourth tadpole alpha-globin mRNA sequence, for which we do not have a cDNA clone, accumulates co-ordinately with the three major mRNA sequences but to a much lower concentration. This pattern of gene expression differs significantly from that of the tadpole beta-globin genes of X. laevis, despite the two classes of genes being closely linked in the genome.     

Sequence organization of two recombinant plasmids containing genes for the major heat shock-induced protein of D. melanogaster.

We have isolated recombinant DNA clones which include cDNA and chromosomal DNA sequences of the major heat shock-inducible gene of Drosophila. With the cDNA fragments used as specific hybridization probes, DNA:DNA reassociation and in situ hybridization analysis demonstrated that the DNA sequences are repeated approximately 7 times in the haploid Drosophila genome, and that gene sequences are present at both the 87A and 87C loci on the cytological map. The cloned cDNA and homologous cloned chromosomal DNA hybridized to mRNA which translated in vitro into the major 70K heat shock-specific protein. Here we summarize a study of the organization of genes coding for the 70K heat shock-specific protein contained in the two recombinant chromosomal DNA plasmids pG3 and pG5. On the basis of R loop hybridization experiments and restriction enzyme analysis, we conclude that a 14 kb fragment, G3, contains three copies of the gene coding for the 70K protein. A second 9.2 kb fragment, G5, contains one copy of the gene coding for the 70K protein. Hybridization of labeled poly(A)-containing RNA to restriction endonuclease-cleaved DNA indicates that the mRNA coding regions in G3 and G5 are each approximately 2100 bp long. The three tandemly repeated genes of G3 are separated by approximately 1400 bp of spacer DNA. The two internal spacer regions in G3 appear to be identical, whereas differences in restriction enzyme sites indicate that the sequences adjacent to the cluster differ from the internal spacer and from each other.     

Isolation and characterization of cDNA clones encoding pathogenesis-related proteins from tobacco mosaic virus infected tobacco plants.

Infection of the tobacco cultivar Samsun NN by tobacco mosaic virus (TMV) results in a hypersensitive response. During this defense reaction several host encoded proteins, known as pathogenesis-related proteins (PR-proteins), are induced. Poly(A)+ RNA from TMV infected tobacco plants was used to construct a cDNA library. Thirty two cDNA clones were isolated and after digestion with different restriction endonucleases, twenty clones were found to code for PR-1a, six clones for PR-1b, and four clones for PR-1c. Two independent cDNA clones of each class were further characterized by DNA sequence analysis. All clones analyzed contained the 138 amino acid coding regions of their respective mature proteins, but only partial sequences of the signal peptides. Minor differences between the nucleotide sequences for clones belonging to the same class were detected. Comparison of the amino acid sequence for PR-1a deduced from its nucleotide sequence with published data obtained by Edman degradation of the protein showed four differences. Analysis of the 3' ends of the cDNA clones indicates that various alternate poly(dA) addition sites are used. Southern blot analysis using these cDNAs as probes suggests the presence of multiple PR-protein genes in the genomes of tobacco and tomato plants.     

Multiplicity and diversity of cloned zein cDNA sequences and their chromosomal localisation

We have constructed and screened cDNA libraries from total maize endosperm poly(A) RNA or from a mRNA fraction enriched in zein sequences. From these libraries we have isolated clones representative of the major classes of zein cDNA sequences and have characterised them by crosshybridisation, by hybrid-selected translation, by in situ hybridisation to maize chromosomes, and hybridisation to genomic Southern blots. We conclude that at least four types of non cross-hybridising zein sequences are present, two coding for light chains and two for heavy chains. At least in the case of the light zeins, there is considerable sequence diversity among the clones which hybridise to each type. Similar results are obtained by translation of the mRNAs selected by each clone. In situ hybridisation shows that the light chain zein genes are located on chromosomes 4, 7, and 10, whilst genes coding for some of the heavy chain zeins are confined to the distal part of the long arm of chromosome 4.