Cloning and expression of a cDNA that comprises part of the gene coding for a spore coat protein of Dictyostelium discoideum.

The three major spore coat proteins of Dictyostelium discoideum are developmentally regulated, cell-type-specific proteins. They are packaged in prespore vesicles and then secreted to form the outer layer of spore coats. We have isolated a cDNA clone from the gene coding for one of these proteins, SP96, a glycoprotein of 96,000 daltons. We screened the cDNA bank by the method of hybrid select translation followed by immunoprecipitation of the translation products with SP96-specific polyclonal antiserum. We found that the gene was first transcribed into stable mRNA a few hours before the time of detection of SP96 synthesis and that the mRNA, like the protein, accumulated specifically in prespore cells and spores. SP96 constituted the same proportion of newly synthesized protein as the proportion of its message in polyadenylated RNA. SP96 appeared to be encoded by a single gene as judged by Southern blot analysis of digested genomic DNA hybridized to the cDNA clone.     

cDNA cloning and chromosomal localization (4q11-13) of a gene for statherin, a regulator of calcium in saliva.

On the basis of the known amino acid sequence of statherin, a human salivary protein, mixed synthetic oligonucleotides were synthesized and used to screen a cDNA library constructed from human parotid-gland mRNA. A cDNA clone coding for statherin was isolated from this library and has been completely sequenced. The cDNA represents a full-length (or nearly full-length) copy of an approximately 640-bp statherin mRNA. Statherin appears to be coded by a single-copy gene that maps to chromosome 4q11-4q13 when somatic-cell hybrids are used.     

Spore coat protein synthesis during development of Dictyostelium discoideum requires a low-molecular-weight inducer and continued multicellularity

The major spore coat proteins of Dictyostelium discoideum are synthesized during the culmination stage of development. In an attempt to examine the regulatory mechanisms involved, spore coat protein synthesis by pseudoplasmodia harvested prior to culmination and incubated in submerged culture under various environmental conditions has been monitored. It is reported that the synthesis of spore coat proteins SP170, SP103, SP94, SP82, SP76, and SP55 is dependent upon the presence of a low-molecular-weight (Mr approx 100), heat-stable factor secreted by cells incubated at high density in buffer. Previous studies have implicated cyclic AMP, ammonia, and amino acids in spore cell differentiation. Partial purification of the spore coat protein inducing factor (SPIF), together with attempts to mimic its activity, indicate that SPIF is not identical with any of these molecules and it is probably also distinct from DIF and "fruit juice," two other factors which regulate the spore-stalk decision and the initiation of culmination, respectively, in D. discoideum. In addition to SPIF, the continued expression of the spore coat protein genes also requires that the integrity of the pseudoplasmodium be maintained. Unlike the expression of many other genes after aggregation, this latter requirement cannot be replaced by exogenous cyclic AMP. Termination of spore coat protein gene expression occurs despite the presence of excess exogenous SPIF and hence involves mechanisms other than the destruction or depletion of SPIF.     

Synthesis of spore proteins during development of Dictyostelium discoideum.

The pattern of synthesis of the spore coat proteins during development of Dictyostelium discoideum has been determined by using immunoprecipitation with spore protein antibody. SP170, SP103, 'SP94', SP82, SP76 and SP55 are all first synthesized just prior to the 'Mexican hat' stage of development (16-18h), but the synthesis of SP72 is delayed. This protein is apparently synthesized as a precursor, P66, which is modified during spore maturation to yield SP72. The nature of the modification is unknown. At their peak period of synthesis during early culmination (18-20h), the spore coat proteins account for 5-9% of total protein synthesis. Shortly after synthesis, these proteins are inserted into the spore coat, where all except SP103 become disulphide-cross-linked during the period 24-30h. SP3 does not accumulate until disulphide-cross-linking of the major spore coat proteins occurs and is itself disulphide-cross-linked into the spore coat. Several additional proteins that are accumulated during development have also been identified, namely P31, P25, P21 and P18. P25 first appears at 18-20h and then continues to be made throughout development. P31 synthesis begins at 12-14h and then largely ceases after approx. 20 h of development. The genes for both P21 and P18 are first expressed early in development, starting at 9-12h. P21 synthesis ceases at approx. 14h, but P18 continues to be synthesized throughout the rest of development. The marked differences in the time period of accumulation of these proteins compared with the co-ordinated syntheses of SP170, SP103, 'SP94', SP82, SP76 and SP55 provide a useful system for analysis of the mechanism of temporal gene expression during development.     

Characterization of a cDNA encoding rat sterol carrier protein2

Sterol carrier protein2 (SCP2) is a 13.2-kD protein that is thought to be involved in the intracellular transport of cholesterol. Using synthetic oligonucleotides based on the protein sequence of SCP2, a clone (SP43) was isolated from a rat liver cDNA library. The DNA sequence revealed that the cDNA could encode a polypeptide of 273 amino acids (28.9 kD) or 143 amino acids (15.3 kD) in which the carboxy-terminal 123 amino acids are identical to the SCP2 protein. RNA blot hybridization revealed that a variety of rat tissues contain a homologous RNA of a size similar to SP43 (approximately 1.5 kb). Levels of SCP2 mRNA increased in parallel with cytochrome P450scc mRNA in the immature gonadotropin-primed rat ovary. The isolation of a cDNA clone encoding SCP2 will facilitate studies on its role in cholesterol metabolism.     

Structure and Expression of a Heat-Shock Protein 83 Gene of Pharbitis nil

Four cDNA clones representing mRNAs whose levels were affected by a photoperiod that induces flowering in Pharbitis nil were isolated by a differential hybridization screening procedure. The level of mRNAs represented by three clones (12L, 15L, and 17L) increased following a photoperiod that induces flowering and that represented by the fourth clone (clone 27) increased under conditions in which flowering was inhibited. DNA sequence analysis showed that one cDNA, clone 17L, is homologous to members of the 83- to 90-kD heat-shock protein (hsp) gene family. The corresponding gene, hsp83A, was isolated and its DNA sequence was determined. hsp83A encodes a protein that exhibits 70% amino acid identity with Drosophila melanogaster HSP83. The P. nil hsp83A gene contains two introns within the coding region. hsp83A mRNA was not detectable in cotyledons of plants grown in continuous light, but its level increased transiently following a 14-h dark period and reached a maximum 2 h after the lights were turned on. A dramatic increase in the level of hsp83A mRNA was also found 2 h after an end-of-day dark treatment. Genomic Southern blot analysis demonstrated that the P. nil hsp83-90 gene family consists of at least six members, one of which appears to be constitutively expressed in the light.     

Cloning of mRNA sequence coding for sex-specific storage protein of Bombyx mori

A major plasma protein, referred to as SP 1, exhibits sex- and stage-specific expression during the larval development of the silkworm, Bombyx mori. We have isolated a plasmid clone bearing a part of mRNA sequence coding for SP 1 and quantitated the amount of SP 1 mRNA by means of RNA blot hybridization using the cDNA probe. The developmental change in the amount of SP 1 mRNA in the fat body closely reflected that of the hemolymph concentration of SP 1, indicating that the biosynthesis of SP 1 is regulated in a sex- and developmental stage-specific fashion at the level of mRNA.     

Studies on hst, a transforming gene which belongs to a new superfamily of growth factors

The hst gene was originally identified in surgically obtained human gastric mucosae as a transforming gene which could transform NIH3T3 cells morphologically. The hst cDNA clone was synthesized from mRNA of one of the NIH3T3 transformants. A human leukocyte genomic library was screened with this cDNA clone, and an hst genomic fragment was obtained. This genomic fragment itself had transforming activity, and the protein coding sequences were proved to be completely identical to those of the cDNA clone prepared from mRNA of the NIH3T3 transformant. This fact suggests that rearrangement or other structural alterations in the coding sequence are not required for the activation of the hst gene. The predicted hst protein consists of 206 amino acids and has a significant homology (40-50%) to fibroblast growth factors and int-2 protein. They together make up a new superfamily of growth factors and transforming genes.     

Molecular cloning, cDNA structure and deduced amino acid sequence for the hormone-induced regulatory subunit (RII beta) of cAMP-dependent protein kinase from rat ovarian granulosa cells

The regulatory subunit of cAMP-dependent protein kinase designated RII beta (RII51) has previously been shown to be the product of a separate gene. This was accomplished by the molecular cloning of a partial cDNA clone estimated to lack 30-45 nucleotides of the 5' end of the coding region. We hereby report the isolation of a cDNA clone for RII beta from rat granulosa cells, extending 43 nucleotides further 5' compared with the previously published cDNA sequence, and from which the entire amino acid sequence (415 residues) of the rat RII beta protein can be deduced. A cAMP regulated mRNA of 3.2 kilobases (kb) for RII beta was detected by the isolated cDNA in rat Sertoli cells.     

Re-expression of 117 antigen, a cell surface glycoprotein of aggregating cells, during terminal differentiation of Dictyostelium discoideum prespore cells

117 antigen is a glycoprotein expressed on the surface of D. discoideum cells at aggregation. It then disappears and is later re-expressed on the surface of a subpopulation of cells at culmination, the terminal differentiation stage (Sadeghi et al. 1987). A cDNA clone was used to show that the appearance of cell surface 117 antigen accurately reflects the expression of the 117 gene as measured by mRNA levels. It was also shown that during multicellular development there is a reciprocal relationship between the levels of 117 mRNA and the mRNA which codes for prespore surface glycoprotein, PsA. Dual parameter flow cytometry was used to demonstrate that the 117 antigen is found on the surface of maturing prespore cells after the PsA glycoprotein disappears, but that it is not found on mature spores. Using three monoclonal antibodies which identify respectively 117 antigen, PsA, and MUD3 antigen (a spore coat glycoprotein--probably Sp96), two new stages of final spore maturation were defined. These results indicate that there is a recapitulation of at least one aggregative cell surface glycoprotein in the prespore subpopulation of cells as they rise up the stalk during final spore development. This raises the possibility that culmination, which involves complex three dimensional morphogenetic movements not unlike those observed during animal embryogenesis, involves components of the two-dimensional pattern seen during aggregation.     

Cloning and characterization of a symbiosis-related gene from an ectomycorrhizal fungus Laccaria bicolor

An in vitro system for a Laccaria bicolor×Pinus resinosa interaction was used to identify and clone a symbiosis-regulated gene from L. bicolor employing the mRNA differential display technique (DDRT–PCR). The DDRT–PCR identified several cDNAs that are differentially expressed as early as 6 h into the interaction. One such cDNA was used to screen a L. bicolor cDNA library enriched for mRNAs expressed during early interaction with red pine seedlings. Characterization of a cDNA clone, PF6.2, showed that it contained a 1551 bp insert coding for a protein of 433 amino acids. Sequence analysis of the PF6.2 cDNA revealed the presence of several evolving repeats in the protein. To confirm this, the gene corresponding to PF6.2 was isolated and sequenced. The PF6.2 gene consisted of seven exons interrupted by six relatively small introns. Although the amino-acid sequence of the PF6.2 did not show significant overall similarity to any previously characterized proteins, of several direct repeats it contained a feature similar to other proteins involved in signal transduction through protein–protein interaction. Northern analysis showed that the PF6.2 mRNA was detectable in the fungus 6 h after interaction and continued to be expressed in established ectomycorrhizas, suggesting that it plays an important role in the formation and maintenance of the symbiosis.     

Isolation and characterization of a cDNA clone encoding rat nucleoside diphosphate kinase

A cDNA clone for cytosolic nucleoside diphosphate (NDP) kinase was isolated from a cDNA library of rat skeletal muscle using synthetic oligonucleotides as probes. The clone constitutes a 621-base pair cDNA sequence including the 456-base pair coding region and 137-base pair 3'-untranslated one with polyadenylation site. The complete primary structure of NDP kinase was deduced from the coding sequence. An NH2-terminal amino acid sequence analysis suggested that the translated enzyme protein suffered proteolytic cleavage followed by modification at the alpha-NH2 group of the newly produced NH2-terminal amino acid residue. Taking this into account, it was tentatively concluded that the mature NDP kinase consists of 147 amino acid residues with a molecular weight of 16,724. Northern blot hybridization analysis showed that NDP kinase mRNA could be detected in total RNA fractions of brain, spleen, heart, lung, liver, kidney, testis as well as skeletal muscle, and that there was no difference in the size of mRNAs from these tissues. Tissue distribution of the mRNA nearly paralleled those of protein moiety and activity of the enzyme.