Alternatively spliced RNAs encode several isoforms of CD46 (MCP), a regulator of complement activation

Five alternative cDNA clones were isolated for CD46, also known as the membrane cofactor protein (MCP) for the factor I-mediated cleavage of the complement convertases. One of these cDNA clones (a) was identical to an earlier MCP clone. The other four CD46 clones 3ontained the four NH₂-terminanl short consensus repeat (SCR) units of MCP, but differed at the region encoding the carboxyl-terminal of the protein which includes an extracellular segment rich in Ser, Thr, and Pro residues, a hydrophobic membrane-spanning domain, and a 33 amino acid cytoplasmic tail. The different CD46 cDNAs have variously: (b) inserted a 93 base pair (bp) exon resulting in a new cytoplasmic tail of 26 amino acids; (c) deleted a 42 bp exon from the extracellular Ser/Thr rich region; (d) used a cryptic splice acceptor sequence to delete 37 bp from an exon encoding transmembrane sequence; or (e) failed to splice the intron after the four SCR units. These were shown by northern blot and polymerase chain reaction to arise by alternative splicing of CD46 RNA. Forms (a), (b), and (c) of CD46 RNA are common in placental RNA, but (d) was rare, and (e) was incompletely processed and therefore aberrant. The polymerase chain reaction (PCR) was used to map the sites of the intron/exon junctions and demonstrate further possible splice variants of CD46. The alternative RNAs for CD46 may correlate to the different isoforms of CD46 found in different tissues, tumors, and in serum.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M58050. Address correspondence and offprint requests to: D. F. J. Purcell.     

Synthesis of cellular and extracellular glycoproteins by cultured human keratinocytes and their response to retinoids

The glycoproteins synthesized by human keratinocytes cultured on 3T3 feeder layers were studied by metabolic labelling. Keratinocytes freed of feeder cells synthesized a complex pattern of cellular and extracellular glycoproteins that was distinct from that of 3T3 cells, dermal fibroblasts and epidermal melanocytes. The effect of low concentrations of all-trans-retinoic acid and arotinoid ethyl ester on glycoprotein synthesis was examined in keratinocyte cultures depleted of vitamin A. Treatment with either retinoid resulted in a 2-3-fold increase in the amount of D-[3H]glucosamine-labelled material in the culture medium. Gel electrophoresis revealed increased incorporation of D-[3H]glucosamine into extracellular glycoproteins of Mr 245,000, 170,000, 140,000, 130,000, 120,000 and 105,000 as well as into glycosaminoglycans in retinoid-treated cultures. The labelling of extracellular glycoproteins with L-[3H]leucine and L-[35S]methionine was also increased by retinoids suggesting increased synthesis of these components rather than an effect on their glycosylation. The Mr 245 000 glycoprotein was identified as keratinocyte-derived fibronectin by immunoblotting, immunoprecipitation and specific binding to gelatin. The results show that retinoids increase the synthesis of glycoprotein as well as glycosaminoglycan components of the extracellular matrix in human keratinocyte cultures. It is suggested that retinoids select for a population of cells that synthesize relatively large amounts of glycosaminoglycan, fibronectin and other as yet unidentified extracellular glycoproteins.     

Secretion of a suppressor cell inducing factor by an interleukin-3 dependent cell line with natural cytotoxic activity

This report describes the morphology, surface markers, growth requirements, and functional activity of the M1-A5 cell line, which was established by the limiting dilution of spleen cells from a mouse bearing a large methylcholanthrene-induced fibrosarcoma. The M1-A5 cells share many of the morphological features of large granular lymphocytes and, in addition, express asialo GM1 and Ly-5 surface markers which are commonly found on natural killer cells (NK) cells. There is no expression of T-cell differentiation antigens, surface immunoglobulin, or the granulocyte/macrophage marker, MAC-1. M1-A5 cells are dependent on exogenous growth factor(s) for survival and will proliferate if cultured in interleukin 3 (IL-3), but not in interleukin 1 (IL-1), interleukin 2 (IL-2), or granulocyte/macrophage colony stimulating factor (GM-CSF). In addition, the M1-A5 cells do not absorb IL-2. Despite their morphology and surface characteristics, the M1-A5 cells do not lyse NK targets such as YAC-1 and RLM1 in 4- or 18-hr cytotoxic assays but do lyse the natural cytotoxic (NC) susceptible target, WEHI-164, and to a very small extent, the M-1 fibrosarcoma cells, in an 18-hr assay. Thus they exhibit NC-like cytotoxic activity. In addition, the M1-A5 cells secrete a small molecular weight factor which activates suppressor cells capable of inhibiting antibody synthesis by cocultured syngeneic spleen cells.     

The population characteristics of howler monkeys (Alouatta caraya) in Northern Argentina

Howler monkeys, Alouatta caraya, were collected in family groups from islands in the Rio Parana in northern Argentina. The body weights, lens weights, dental age and reproductive status of 261 individuals illustrated sexually dimorphic body weights after a period of accommodation as young adults, a large proportion of older females (even though there are more young adult males than females) and a higher percentage of pregnancies among these older females. Seventeen families, representing the entire social system of one island, showed that about three quarters were adult (equally divided between the sexes but with fewer older males than females) and the remaining quarter were immature (with an equal sex ratio). Half of the adult females were either pregnant or suckling young. The mean family size is seven which includes peripheral males. Group size and composition as well as adult to immature proportions compared closely with data on A. caraya in the Chaco of Argentina and the 1951 census of A. palliata on Barro Colorado Island. It can be said that when these two species of Alouatta occur in small size families, they have in common: a high proportion of adults to immature, a low proportion of infants, and can have either an equal sex ratio (A. caraya) or preponderance of females (A. palliata).     

Single base mutation in the type III procollagen gene that converts the codon for glycine 883 to aspartate in a mild variant of Ehlers-Danlos syndrome IV

Experiments were carried out to test the hypothesis that a 19-year-old proband with a mild variant of Ehlers-Danlos syndrome type IV had a mutation in the gene for type III procollagen. cDNA and genomic DNA were analyzed by using the polymerase chain reaction and cloning of the products into M13 filamentous phage. A mutation was found that converted the codon for glycine 883 of the triple-helical domain in one allele for type III procollagen to a codon for aspartate. The polymerase chain reaction introduced a few artifactual single base substitutions. Also, it was difficult to distinguish copies from the two alleles in many of the M13 clones. Therefore, several different strategies and analyses of about 50,000 nucleotide sequences in a series of clones were used to demonstrate that the mutation in the codon for glycine 883 was the only mutation in coding sequences for the triple-helical domain of type III procollagen that could have contributed to the phenotype. The same mutation in the codon for glycine 883 in one allele for type III procollagen was found in the proband's 52-year-old father who also had a mild variant of Ehlers-Danlos syndrome type IV. The type III procollagen synthesized by the proband's fibroblasts was analyzed by polyacrylamide gel electrophoresis. Less type III procollagen was secreted by the proband's fibroblasts than by control fibroblasts. Also, the thermal stability of the type III procollagen synthesized by the proband's fibroblasts was lower than the thermal stability of normal type III procollagen as assayed by brief protease digestion. The results, therefore, demonstrated that the single base mutation that converted the codon of glycine 883 to a codon for aspartate destabilized the entire triple helix of type III procollagen and probably accounted for the mild phenotype of Ehlers-Danlos syndrome type IV seen in the proband and her father.