Abstract
A polymerase chain reaction-based cloning approach was employed in order to identify ADP-ribosylation factors (ARF) in murine 3T3-L1 cells and to study their expression before and after differentiation of cells to the adipocyte-like phenotype. Partial sequences comprising the effector domains of ARF were amplified with degenerate primers and cloned. Five of these sequences were identified as murine homologues of known human ADP-ribosylation factors (ARF 1, 2, 4, 5, and 6). In addition, partial sequences of two previously unknown isoforms were found, and complete cDNA clones were isolated from a rat fat cell library and were sequenced. Both sequences harbor a putative myristoylation site in position 2, the known consensus sequences presumably involved in GTP binding and hydrolysis, and lack cysteine residues in the C terminus. Their amino acid sequences share a 56 and 41% identity, respectively, with human ARF 1. Based on a comparison with the known ARF isoforms, the first clone appears to represent the mammalian homologue of a known sequence from Drosophila (dARL 1, 79% identity) and was therefore designated rARL 1. The second clone resembled none of the known ARF-like proteins and was designated rARL 4. mRNA of ARL 4 was undetectable in the fibroblasts but abundant in the adipocyte-like phenotype, its expression starting on day 6 of the differentiation. In contrast, ARF 1, 2, and 5 were unaltered by differentiation of the 3T3-L1 cells; mRNA levels of ARF 6, and also of ARL 1 and ARF 4, were reduced after differentiation. It is suggested that the function of ARL 4 is related to the adipocyte-like phenotype of 3T3-L1 cells.
Highlights
In the adipocyte-like phenotype,its expression starting binding proteins. They have been identified as cofactors reon day 6 of the differentiation.In contrast, ADP-ribosylation factors (ARF) 1,2, and quired for the cholera toxin-catalyzed ADP-ribosylation ofG, 5 were unaltered by differentiation of the 3T3-Ll cells; mRNA levels of AFW 6, and of ARL 1andARF 4, were (Kahn and Gilman1,984)
PCR Cloning of A R F Zsoforms-Degenerate oligonucleotide primers matching the domainsPM1 and PM3 (see Fig. 4; nomenclature according to Valencia et al (1991)) were employed to amplify a domain harboring theeffector loop ofARF proteins
141 150 test a possible involvement of ARF proteins in insulin-dependent glucose transport, we studied mRNA levels of ARF isoforms in the major glucose-utilizing tissues from normal and strep
Summary
Wendlingweg 2, D-52057Aachen, Germany. “el.: 49-241-8089120; Fax: phate);ARF, ADP-ribosylation factors; ARL, ADP-ribosylation factor-. Like; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s). Library Screeningand DNA Sequencing-PCR products subclonedin pUCl8 were isolated with restriction enzymes EcoRI and BamHI and used as probes to screen a rat fat cell A - g t l l cDNA library (RLlOllb; Clontech, Palo Alto, CA). Probes were generated with the Klenow fragment of DNA polymerase I and [32P]CTP fromthe 150-bpPCR products (ARF 1,2, 4, 5, and 6) or full-lengthcDNA probes (ARL 1and 4) by random oligonucleotidepriming (Feinberg and Vogelstein, 1983). The nylon membranes were hybridized a t 42"C, and blots hybridized with the PCR products were washed three times at 55 "C in 0.12 M NaC1, 0.012 M sodium citrate, 0.1% SDS; blots hybridized with full-length cDNA were washed twice with 0.12 M NaC1,0.012M sodium citrate, 0.1%SDS and once with 0.015 M NaCl, 0.0015 M sodium citrate, 0.1% SDS.In control experiments, all blots were hybridized with an actin probe in order t o ascertain that equal amounts of mRNA were present in comparable samples
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