Molecular cloning of cDNAs for the nerve-cell specific phosphoprotein, synapsin I.

To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated from rat brain. Synapsin I mRNA was specifically enriched by immunoadsorption of polysomes prepared from the brains of 10-14 day old rats. Employing this enriched mRNA, a cDNA library was constructed in pBR322 and screened by differential colony hybridization with single-stranded cDNA probes made from synapsin I mRNA and total polysomal poly(A)+ RNA. This screening procedure proved to be highly selective. Five independent recombinant plasmids which exhibited distinctly stronger hybridization with the synapsin I probe were characterized further by restriction mapping. All of the cDNA inserts gave restriction enzyme digestion patterns which could be aligned. In addition, some of the cDNA inserts were shown to contain poly(dA) sequences. Final identification of synapsin I cDNA clones relied on the ability of the cDNA inserts to hybridize specifically to synapsin I mRNA. Several plasmids were tested by positive hybridization selection. They specifically selected synapsin I mRNA which was identified by in vitro translation and immunoprecipitation of the translation products. The established cDNA clones were used for a blot-hybridization analysis of synapsin I mRNA. A fragment (1600 bases) from the longest cDNA clone hybridized with two discrete RNA species 5800 and 4500 bases long, in polyadenylated RNA from rat brain and PC12 cells. No hybridization was detected to RNA from rat liver, skeletal muscle or cardiac muscle.     

Alkaline phosphatase conjugated protein A as a sensitive reagent to immunoscreen an expression cDNA plasmid library: isolation of cDNA to the calcium-binding protein of the chick embryonic chorioallantoic membrane

A highly efficient immunoscreening procedure has been developed to isolate cDNA clones to the calcium-binding protein (CaBP) of the chick embryonic chorioallantoic membrane (CAM). A library of total CAM cDNA was constructed using the expression plasmid vector, pUC 19. Bacterial clones containing plasmids with CaBP cDNA inserts were detected immunohistochemically based on their expression of hybrid CaBP protein sequences. For immunodetection, nitrocellulose bacterial colony replicas were treated with specific antibodies to the CaBP followed by incubation with Staphylococcus aureus Protein A conjugated with alkaline phosphatase (AP) which served as a secondary immunoreagent. Positive clones were then histochemically identified based on AP enzyme activity. The identity of the immunopositive clones was further verified by in vitro translation of mRNA selected by hybridization to the cloned cDNA. The AP-based immunoscreening procedure yields stable reaction products with relatively low background, and should find general application for isolating specific cDNA clones from expression cDNA libraries.     

Identification of a set of genes expressed during the G0/G1 transition of cultured mouse cells.

To identify previously undetected genes that may be involved in the transition from a resting state (G0) to a proliferative state (G1) of mammalian cells, we set out to isolate cDNA clones derived from mRNAs that appear in serum-stimulated cells in the absence of protein synthesis. A lambda cDNA library was prepared using poly(A)+ RNA from BALB/c 3T3 cells that had been brought to quiescence and subsequently stimulated with serum in the presence of cycloheximide. Approximately 50 000 recombinant phage plaques were screened, and 357 clones were isolated that hybridized to probes derived from stimulated-cell RNA but not to probes from resting-cell RNA. Cross hybridization analysis showed that four RNA sequence families account for approximately 90% of these clones. One of the clones hybridized to an actin probe; none hybridized to any of 13 oncogene probes tested. Five different RNAs that appear to be previously uncharacterized have been further analyzed. These RNAs accumulate and decay rapidly following stimulation by serum or purified growth factors, or by a tumor promoter, and they are superinduced by serum in the presence of cycloheximide. Three of the RNAs could be enriched by hybridization to cDNAs and translated in vitro, yielding proteins of approximately 43, 40 and 35 kd, respectively.     

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.