Wim Quax

THE STRUCTURE OF THE VIMENTIN GENE

The structure of the chromosomal gene encoding the intermediate filament protein vimentin is described. This gene, which is present as a single copy in the hamster genome, comprises about 10 kb of DNA and contains more than 80% of intron sequences. S1 mapping and sequence analysis reveal nine exons with a total length of 1848 nucleotides. For the complete primary structure of hamster vimentin, 464 amino acids are predicted, giving a molecular weight of 53,500 daltons. The intron positions are at codons 186, 206/207, 238/239, 292/293, 334/335, 408, 423, and 451/452. The overall homology with chicken desmin is 60% and is even higher in the central (alpha-helical) regions of both molecules. Cross-hybridization at the DNA level, however, is low. Comparison of the amino acid sequence of vimentin with prekeratin sequences shows that there is lesser homology of primary structure, but both the position and size of alpha-helical regions are strongly conserved. At the 5 end of the gene there is a consensus promoter sequence. The first AUG start codon is found 132 nucleotides downstream of the estimated cap site. The 3 nontranslated sequence shows homologies with the chicken vimentin gene. An interesting feature of the vimentin gene is a stretch of 44 nucleotides of alternating dC and dA within intron 2 that may form left-handed Z-DNA.

The genes coding for the cytoskeletal proteins actin and vimentin in warm-blooded vertebrates.

Recombinant plasmids were made containing cDNAs synthesized on hamster mRNAs coding for cytoskeletal (beta‐ or gamma‐) actins and for vimentin. Hybridization of the actin probe on restriction digests of one avian and five mammalian DNAs yielded multiple bands; the vimentin probe revealed only one band (accompanied by 2‐3 faint bands in some DNAs). The results obtained with the vimentin probe indicate that the corresponding coding sequences: (a) are highly conserved in warm‐blooded vertebrates like the actin sequences; (b) have strongly diverged from those coding for other intermediate filament proteins, since hybridization of the vimentin probe does not lead to a diagnostic multiband pattern; and (c) most likely contribute to single gene, in contrast to the sequences coding for other cytoskeletal proteins. Hybridization of the probes on mRNAs from the different sources used showed that the non‐coding sequences of both vimentin and actin genes are conserved in length.

Characterization of the hamster desmin gene: Expression and formation of desmin filaments in nonmuscle cells after gene transfer

The structural organization of the hamster gene encoding the intermediate filament (IF) protein desmin has been determined. The gene, 6.5 kb in length, contains nine exons with a total length of 2169 nucleotides. Remarkably, the intervening sequences map at positions that fully correspond to those of the vimentin gene. The derived complete primary structure for hamster desmin (468 amino acids; 53,250 daltons) reveals striking species variations in the NH2-terminal domain of desmin. A plasmid containing the complete transcription unit of the desmin gene was transfected into hamster lens cells and into human epithelial (HeLa) cells. In both nonmuscle cell lines the desmin gene was biologically active. The synthesized desmin assembled into authentic IFs, as monitored by immunofluorescence. Double immunofluorescence staining showed that the newly formed desmin filaments colocalize with preexisting vimentin filaments, but not with preexisting keratin filaments.

BOVINE BETA-CRYSTALLIN COMPLEMENTARY-DNA CLONES - ALTERNATING PROLINE ALANINE SEQUENCE OF BETA-B1 SUBUNIT ORIGINATES FROM A REPETITIVE DNA-SEQUENCE

A library of recombinant plasmids carrying complementary DNA sequences synthesized from bovine lens messenger RNAs was constructed. Clones coding for five different beta-crystallin subunits: beta B1, beta B3, beta Bp, beta s, beta A3 (and beta A1), were identified by means of hybridization selection, followed by one- and two-dimensional gel electrophoresis of the translational products. Under rather stringent conditions each of these clones hybridizes with its corresponding mRNA and does not show significant cross-hybridization with mRNAs coding for other beta-crystallins, except in the case of the homologous beta A3 and beta A1-crystallins. The beta A3 and beta A1 subunits seem to be encoded by one mRNA using two different AUG codons as start position for translation. We have also determined the nucleotide sequence of a beta B1-crystallin cDNA (pBL beta B1) which enabled us to deduce the complete amino acid sequence of the protein. The beta B1-crystallin, a characteristic component of the high molecular weight crystallin aggregate (beta H), is internally homologous both at DNA and protein level as has been reported for gamma- and other beta-crystallins. This is in agreement with the idea that these proteins had a common ancestral precursor gene that internally duplicated. The G + C content of the coding sequence of beta B1 is very high: 67% overall and even 84.2% for the first 170 nucleotides, due to a remarkable non-random codon usage. A proline/alanine repetition in the N-terminal domain of the protein is encoded by a repetitive simple DNA sequence.

Complete structure of the hamster alpha A crystallin gene. Reflection of an evolutionary history by means of exon shuffling.

The eye lens contains a structural protein, alpha crystallin, composed of two homologous primary gene products alpha A2 and alpha B2. In certain rodents, still another alpha crystallin polypeptide, alpha AIns, occurs, which is identical to alpha A2 except that it contains an insertion peptide between residues 63 and 64. In this paper we describe the complete alpha A crystallin gene that has been cloned from DNA isolated from Syrian golden hamster. Evidence is provided that the alpha A gene is present as a single copy in the hamster genome. The detailed organization of the gene has been established by means of DNA sequence analysis and S1 nuclease mapping, revealing that the gene consists of four exons. The first exon contains the information for the 68 base-pair long 5 non-coding region as well as the coding information for the first 63 amino acids. The second exon encodes the 23 amino acid insertion sequence, the third exon codes for amino acid 87 to 127 of the alpha AIns chain, whereas the last exon encodes the C-terminal 69 amino acids and contains the information for the 523 base-pair long 3 non-coding region. The second exon is bordered by a 3 splice junction (A X G/G X C), which deviates from the consensus for donor splice sites (A X G/G X T). This deviation is found in both hamster and mouse. An internal duplication was detected in the first exon by using a DIAGON-generated matrix for comparison. By means of similar DIAGON-generated matrices it was confirmed that the amino acids coded for by the third and fourth exons are homologous to the small heat-shock proteins of Drosophila, Caenorhabditis and soyabean. The implications of the differential splicing and the evolutionary aspects of the detected homologies are discussed.

The human desmin and vimentin genes are located on different chromosomes

We have used somatic cell hybrids of Chinese hamster X man and mouse X man to localize the genes (des and vim) encoding the intermediate filaments desmin and vimentin in the human genome. Southern blots of DNA prepared from each cell line were screened with hamster cDNA probes specific for des and vim genes, respectively. The single-copy human des gene is located on chromosome 2, and the single-copy human vim gene is assigned to chromosome 10. Partial restriction maps of the two human genomic loci are presented. A possible correlation of the des locus with several reported hereditary myopathies is discussed.

Vimentin and Desmin cDNA Clones: Structural Aspects of Corresponding Proteins and Genes

Microtubules, microfilaments, and intermediate-sized filaments (IF) are the major cytoskeletal elements of most eukaryotic cells. The function of the IF is far from being fully understood. Maintenance of cell shape and/or anchorage to the substratum have been suggested to represent at least some of the functions of these fibrous structures.’ Identification and visualization of the spatial orientation in intact cells have been achieved by electron micro~copy~’~ and immunofluorescence microscopy.4~’ Mainly from the latter studies it appeared that as a rule IF proteins are tissue-specific. For instance mesenchymal cells comprise vimentin as subunit of their IF, whereas cells from an epithelial source contain cytokeratins. An exception of the aforementioned regularity is the vertebrate lens that due to its epithelial origin should produce prekeratin. Nevertheless, only vimentin is found and none of the other IF protein subunits! The reason for deviation from the “rule” is not clear yet. In view of the observation that cells from different embryonic origin start synthesizing vimentin as soon as they are subjected to culturing, it might well be that the eye lens, due to its peculiar growth pattern, arising from a monolayer of epithelial cells that are attached to a natural substratum (the lenticular capsule),’ resembles to some extent cells growing in culture (FIGURE 1). This example of regulation of IF gene expression led us to undertake investigations upon the molecular basis of this process. In order to facilitate comparative studies we performed molecular cloning of both vimentin and desmin cDNAs. The isolated clones served primarily a threefold aim: The determination via the nucleotide sequence of the primary structure of the proteins; the prediction of their secondary structure contained in a general model, presumably valid for all IF protein subunits; and the isolation of the corresponding genes from genomic DNA. Moreover, the isolated genes whose structures have been determined will be used for expression studies in heterologous cell systems.

Evolution of the single copyαA-crystallin gene: differently sized mRNAs of mammals and birds show homology in their 3′ non-coding regions

AbstractαA-Crystallin, a major structural polypeptide of the vertebrate eye lens, is evolutionarily highly conserved. We have analyzed the corresponding nucleic acid sequences in both genomic DNA digests as well as in lens cytoplasmic RNA preparations from a wide variety of vertebrates by blot hybridization with cloned rat αA2-crystallin cDNA probes. The probes are not able to hybridize under any conditions to RNA and DNA derived from fishes and amphibia, but do show substantial homology with the sequences of mammals, birds and reptiles. The αA-crystallin gene, which has been isolated from a hamster gene library occurs only once in the haploid genome. Coding and 3′-untranslated regions of αA2-crystallin mRNA are conserved among all mammals and birds examined. However, the regions comprising the conserved sequences are differently represented in the ultimate mRNA. The αA2-mRNA 3′-non-coding regions of reptiles and birds are 300–550 bases longer than those of mammals. Some rodents produce next to the αA2-mRNA another messenger that encodes the αAlns-polypeptide possessing an insertion of 22/23 amino acid residues between positions 63 and 64 of the αA2-polypeptide chain. αA2 and αAlns-mRNA are generated from a single gene as major and minor species, respectively, in a proportion which is similar to the ratio of the polypeptides foundin vivo andin vitro. The size heterogeneity of the αA2-mRNA from most mammals examined is due to the variable size of the poly(A) tail.

Organization and Expression of the Vimentin and Desmin Genes

The detailed view of the electron microscope has made it possible to identify a number of filamentous structures in the eukaryotic cell. Differences in general morphology and diameter, in addition to the susceptibility to certain drugs, have been used to classify the observed cytoskeletal filaments. Actin filaments (6 nm in diameter), microtubules (25 nm) in nonmuscle cells, and myosin filaments (15 nm) in muscle cells were readily characterized. However, the filaments with a diameter of 9–10 nm (intermediate between the diameter of actin-containing microfilaments and those of myosin containing thick filaments and microtubules) were initially not regarded as a different class of fibers.