Regulation of the gene encoding the p24 actin-binding protein in Dictyostelium discoideum.

We have isolated cDNA clones on the basis of sequence similarity to the gene encoding the cyclic cAMP-binding protein CABP1 of Dictyostelium discoideum. The predicted amino acid sequence of the cloned cDNAs shows that the homology to CABP1 is restricted to a region rich in proline, glycine, glutamine, and tyrosine. Sequence comparison indicates that the cloned cDNAs encode the actin-binding protein p24. We have examined by RNA blot hybridization the expression of the gene encoding p24. For cells developed in suspension, the levels of p24 mRNA increase rapidly during early development, reaching a peak at 3-4 h. Addition of high concentrations of exogenous cAMP during the first 4 h of development produced little or no effect on the accumulation of p24 mRNA. Treatment with cAMP during subsequent stages of development reduced the levels of p24 mRNA. We attempted to determine if the synthesis of new proteins during early development is a requirement for the reduction in p24 mRNA levels by treating the cells with protein synthesis inhibitor. Unexpectedly, the addition of the inhibitor cycloheximide resulted in an increase in the level of p24 mRNA. The roles of cycloheximide and cAMP on the expression of the p24 gene are discussed.     

Isolation of cDNAs coding for epitopes shared by microtubule-associated proteins and neurofibrillary tangle in Alzheimer's disease

5 cross-hybridizing cDNA clones of sizes 2.2 (3 cDNAs), 1.3 and 0.8 kb corresponding to tau microtubule-associated protein have been isolated from a rat brain lambda gt11 expression library. Antibodies affinity-purified using the fusion protein encoded by the cDNAs were observed to label tau polypeptides on Western blots and Alzheimer's neurofibrillary tangles.     

Multiple chick tropoelastin mRNAs

Several overlapping chick tropoelastin cDNAs were isolated from a lambda gt11 cDNA library constructed from whole 10 day chick embryo total RNA. Comparison of the nucleotide sequence of the 2.3 kb tropoelastin cDNA to the sequences published by Bressan et al. (1) and Tokimitsu et al. (2) revealed the presence of two inserts (72 and 30 base pairs) in the cDNA derived from embryonic tissue. Northern blot analysis of 14 day embryonic aortae RNA with tropoelastin cDNA clones showed hybridization to a 3.5 kb mRNA. However, S1 nuclease protection experiments performed on RNA extracted from the same tissue showed that at least two if not more tropoelastin mRNAs exist and that the proportion of each varies in the ages examined. These results provide an origin and substantiate the differential expression of the multiple tropoelastin polypeptides found in developing chick aortic tissue.     

Differential accumulation of potato tuber mRNAs during the hypersensitive response induced by arachidonic acid elicitor

Accumulation of messenger RNAs in potato tuber discs was analysed during the hypersensitive response induced by treatment with the biotic elicitor arachidonic acid. In vitro translation of polysomal poly(A)⁺ RNAs indicated that the accumulation of some sixteen mRNAs varied following treatment with arachidonic acid, and that the level of thirteen of these was increased. Two cDNA closes (pSTH-1 and-2) were isolated from a library of elicitor-treated tissue cDNAs. Northern blot analysis using these clones as molecular probes indicated that the levels of at least two mRNAs were markedly increased after elicitor treatment. In hybrid-released translation experiments, each of the cDNA clones selected more than one mRNA. Translation of these mRNAs yielded two polypeptides of M_r 45 000 (for the pSTH-1 clone), and three polypeptides of M_e 17 000 (for the pSTH-2 clone). The low molecular weight polypeptides may correspond to potato pathogenesis-related (PR) proteins.     

Induction of thaumatin‐like proteins (TLPs) in Rhizoctonia solani‐infected rice and characterization of two new cDNA clones

Thaumatin‐like proteins (TLPs) were shown to be induced in rice plants (cv. IR58) that were infected with the sheath blight fungus, Rhizoctonia solani . Western blot analysis revealed the presence of two TLPs with sizes of 25 and 24 kDa which are different from a previously reported TLP with a size of 15.6 kDa from rice plants infiltrated with the non‐pathogenic bacterium, Pseudomonas syringae pv. syringae . By probing a cDNA expression library prepared from RNA isolated from R. solani ‐infected rice plants with a TLP antibody, several putative TLP cDNA clones were isolated and sequenced. The cDNA clones appeared to be derived from two different genes which shared only 77% sequence identity with each other and a lower percentage of sequence identity with the previously reported TLP cDNA clone. Southern blot analysis with the two TLP cDNAs revealed different rice genomic DNA fragments. Northern blot analysis also confirmed that a 1.1‐kb RNA detectable by the TLP cDNA inserts was induced by fungal infection. Thus rice TLPs are encoded by a family of at least three genes which are differentially expressed in responses to bacterial or fungal pathogens.     

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.     

Prediction of the coding sequences of mouse homologues of KIAA gene: III. the complete nucleotide sequences of 500 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have conducted a human cDNA project to predict protein-coding sequences (CDSs) in large cDNAs (> 4 kb) since 1994, and the number of newly identified genes, known as KIAA genes, already exceeds 2000. The ultimate goal of this project is to clarify the physiological functions of the proteins encoded by KIAA genes. To this end, the project has recently been expanded to include isolation and characterization of mouse KIAA-counterpart genes. We herein present the entire sequences and the chromosome loci of 500 mKIAA cDNA clones and 13 novel cDNA clones that were incidentally identified during this project. The average size of the 513 cDNA sequences reached 4.3 kb and that of the deduced amino acid sequences from these cDNAs was 816 amino acid residues. By comparison of the predicted CDSs between mouse and human KIAAs, 12 mKIAA cDNA clones were assumed to be differently spliced isoforms of the human cDNA clones. The comparison of mouse and human sequences also revealed that four pairs of human KIAA cDNAs are derived from single genes. Notably, a homology search against the public database indicated that 4 out of 13 novel cDNA clones were homologous to the disease-related genes.     

Cloning and characterization of a lectin from the octocoral Sinularia lochmodes

In the present study, the entire amino acid sequence and cDNA structure encoding the d-galactose-binding lectin, SLL-2, isolated from the octocoral Sinularia lochmodes, were determined. SLL-2 regulates the morphology of symbiotic dinoflagellates Symbiodinium spp. through unknown mechanisms. Here, three cDNAs that encode SLL-2 were cloned and characterized. All the SLL-2 cDNAs encoded 142 amino acids with high similarity to each other. The mature subunit of SLL-2 was found to be composed of 94 amino acids and to contain one putative glycosylation site common to all three SLL-2. N-Glycopeptidase F treatment of SLL-2 resulted in a protein band shift from 16.5 to 9.5kDa in SDS-PAGE, confirming that SLL-2s are glycoproteins. Two-dimensional polyacrylamide gel electrophoresis analysis of the deglycosylated SLL-2 indicated a presence of three polypeptides as encoded in SLL-2 cDNAs. The deduced sequences of SLL-2 cDNAs had a similarity to the C-terminal region of discoidin I, the slime mold Dictyostelium discoideum lectin.     

The glucosinolate-degrading enzyme myrosinase in Brassicaceae is encoded by a gene family

A full-length cDNA clone (MB3) and three partial clones (MA1, MB1 and MB2) which encode myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) were isolated from a Sinapis alba (white mustard) cDNA library. Nucleotide sequence analysis of these clones revealed that they are encoded by a gene family. Southern blot analysis with gene-specific probes showed that the gene family consists of a least two subfamilies (MA and MB) each with several members both in S. alba and in Brassica napus (oilseed rape). In Arabidopsis thaliana (wall cress) only three myrosinase genes seem to be present. Northern blot analysis indicated that all the myrosinase mRNA species have the same size, approximately 1.95 kb.     

The cDNA of the two isoforms of bovine cGMP-dependent protein kinase

cDNAs encoding the isoform I alpha of the cGMP-dependent protein kinase were isolated from a bovine trachea smooth muscle cDNA library constructed in lambda gt10. The deduced protein sequence is identical with the protein sequence obtained by Edman degradation of the bovine lung enzyme [(1984) Biochemistry 23, 4207-4218]. Alternate cDNA clones were isolated which code for a protein slightly different within the aminoterminal part from the known amino acid sequence. These alternate cDNAs contain the sequence of a peptide identified in the isoform I beta of cGMP-dependent protein kinase. Northern blot analysis of poly(A)+ RNA from bovine trachea smooth muscle indicated the presence of two different mRNA species of about 6.2 kb.     

Isolation and characterization of a cdc 2 cDNA from Dictyostelium discoideum.

A cdc2 homologous sequence was amplified from Dictyostelium discoideum by the polymerase chain reaction and used to isolate several cDNA clones. The amino acid sequence encoded by these cDNAs exhibited approx. 60% identity to the Cdc2 proteins of other species. A cDNA containing the entire coding sequence complemented the temperature sensitive cdc28 mutant of Saccharomyces cerevisiae, although growth of the transformants was slow and limited. Southern blot analysis of restriction digests under high stringency conditions provided evidence that Dictyostelium contains a single cdc2 gene, although at lower stringency multiple fragments were detected, suggesting the existence of a cdc2 gene family. Northern blot analysis of RNA from different stages of Dictyostelium development showed that cdc2 mRNA levels increased during aggregation and then decreased to low levels by the pseudoplasmodial stage of development. By contrast, cdc2 mRNA levels remained relatively constant as cells passed from exponential growth to the stationary phase.     

Isolation and nucleotide sequences of cDNA clones encoding ADP-glucose pyrophosphorylase polypeptides from wheat leaf and endosperm

A cDNA clone (WL : AGA.1) encoding wheat leaf ADP-glucose pyrophosphorylase has been isolated from a gt11 expression library, by immunological screening with anti-spinach leaf ADP-glucose pyrophosphorylase serum. The WL : AGA.1 cDNA is 948 bp long and contains approximately 55% of the complete wheat leaf ADP-glucose pyrophosphorylase mRNA sequence, estimated from Northern blot experiments. A wheat endosperm cDNA library was subsequently constructed in gt11 and six clones hybridising to the cDNA insert of clone WL : AGA.1 were isolated. The longest of these wheat endosperm ADP-glucose pyrophosphorylase cDNAs, clone WE : AGA.7, is nearly full-length (1798 bp), indicated by Northern blot analysis of wheat endosperm mRNA and nucleotide sequence analysis.Southern hybridisation analysis and restriction enzyme mapping indicated that the wheat leaf and wheat endosperm ADP-glucose pyrophosphorylase cDNAs and genes are members of two distinct gene families. In addition, restriction enzyme mapping revealed polymorphism in the wheat endosperm ADP-glucose pyrophosphorylase cDNAs, indicating the existence of at least two wheat endosperm ADP-glucose pyrophosphorylase gene sub-families.Subsequent nucleotide sequence analysis indicates that there is approximately 55% identity between wheat leaf and wheat endosperm ADP-glucose pyrophosphorylase cDNAs. In contrast, members of each sub-family of endosperm cDNA, represented by clones WE : AGA.3 and WE : AGA.7, are 96% identical.