The interactions between Ro and La proteins and hY RNAs have been analysed. The binding site for the 60 kDa Ro protein on hY RNAs is shown to be the terminal part of the base paired stem structure, which contains the most highly conserved sequence among hY RNAs. The bulged C-residue within this region plays an important role in the recognition by this protein. The same regions of hY RNAs are essential for the association of the 52 kDa Ro protein with the RNAs, strongly suggesting that the 60 kDa Ro protein is required for the 52 kDa Ro protein to bind, presumably via protein-protein interactions, to Ro RNPs. The binding site for the La protein on hY RNAs is shown to be the oligouridylate stretch near the 3'-end of the RNAs, which is also recognized when additional nucleotides flank this motif at the 3'-side. Additional sequence elements in hY3 and hY5, but not in hY1, are bound by the La protein as well. Deletion mutagenesis showed that the RNP motif, previously identified in many ribonucleoprotein (RNP) proteins and in some cases shown to be almost sufficient for the interaction with RNA, of both the 60 kDa Ro and the La protein are not sufficient for the interaction with hY RNAs. Substantial parts of these proteins flanking the RNP motif are needed as well. It is likely that they stabilize the correct conformation of the RNP motif for RNA binding.

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Varying lengths of the hamster desmin and vimentin promoter regions were fused to the bacterial chloramphenicol acetyl‐transferase gene. These constructs were transfected into two different myogenic cell lines, T984 and C2C12. In both cell lines an increase in endogenous desmin expression takes place upon myogenesis. A region between −89 and +25 bp relative to the desmin transcription initiation site directs high‐level tissue‐ and stage‐specific expression upon in vitro myogenesis. At the myoblast stage, C2C12 cells appeared to express both desmin and vimentin, whereas in T984 myoblasts only vimentin expression was detected. Although vimentin is expressed during all stages of myogenesis, a strong decrease in vimentin expression occurs during differentiation of C2C12 cells. Vimentin–CAT constructs followed the endogenous expression pattern, showing that this down‐regulation is mediated by 5′ flanking sequences. Vimentin promoter activity is modulated by at least two separate regions, both in myogenic and in non‐myogenic cell lines.

SUMMARYThe simultaneous deleclion of anti-La. anti-60-kD Ro and anli-52-k[5 Ro anlibodies by immunobloUing is greatly improved by changing the crosslinking level in the gel lo an aerylamide/ bisacrylamide ratio of 19:1. Using this method Tor the analysis of a number of systemie lupus erythemalosus (SLE) and Sjogren's syndrome patienl sera il was observed thai antibody to the 52-kD Ro protein without anii-60-kD Ro antibody was restrieted to Sjogren's syndrome patients (9/26). whereas antibody to tlie 60-kD Ro protein without contaminating anti-52-kD Ro antibody was only found in SLE patients (8/38). Moreover, in Sjogren's sydrome patient sera anti-Ro antibody was found only in eombination with anti-La antibody (20/26), whereas in SLE patienl sera anti-Ro antibody eould be found without detectable anti-La speeifieity (4/38). Double immunofluoreseenee mieroseopy revealed that the 52-kD Ro and lhe 60-kD Ro proteins eo-locali/e in the cytoplasm as well as in the nucleus, whereas immunoprecipitalion of f'-P]-labelled HeLa cell extract wiih monospecific anti-52-kD Ro and anli-60-kD Ro sera showed that bdlh proteins are assoeiated wilh lhe Ro RNAs, These dala suggest the presenee of boUi lhe 52-kD and lhe 60-kD Ro proteins in the same ribonueleoproiein eomplexes. To study the evolutionary conservation ofthe 52-kD Ro. the 60-kD Ro and the La proteins, e.xiracts of eell lines derived from various mammalian speeies were analysed on Western blots using monospecitic human antibodies. In eontrast lo lhe(il)-kD Roand the La aniigens whieh are well conserved in evolution, the 52-kD Ro antigen could be detected in primate eells only by this immunologieal approach.