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.     

Coding sequence and growth regulation of the human vimentin gene.

We have established the complete coding sequence of the human vimentin gene. It had 91% homology to the coding sequence of the Syrian hamster vimentin gene (Quax et al., Cell 35:215-223, 1983) and partial homology to several other sequences coding for intermediate filament proteins. The most striking difference between the Syrian hamster and human vimentin genes was in the 3' untranslated region, which was considerably longer in the Syrian hamster. Using RNA blots and a human vimentin cDNA clone from an Okayama-Berg library, we have established that expression of the vimentin gene was growth regulated. The steady-state levels of cytoplasmic vimentin mRNA in 3T3 cells were increased by serum and platelet-derived growth factor, but not by epidermal growth factor, insulin, or platelet-poor plasma. The increase in expression of the vimentin gene that occurred when G0-phase cells were stimulated to proliferate was detected in six different cell types from four different species. The expression of the vimentin gene was also increased when HL60 cells were induced to differentiate by phorbol esters; it decreased when differentiation was induced by retinoic acid.     

Developmental expression of a neurofilament-M and two vimentin-like genes in Xenopus laevis

A hamster vimentin cDNA probe has been used to isolate and characterize three Xenopus laevis intermediate filament genes, named XIF1, XIF3 and XIF6. Of these, XIF6 shows 89% homology at the amino acid level to a portion of porcine neurofilament-M. XIF6 is transcribed solely in nervous tissue of embryos, commencing at the late neural tube stage. Expression is totally dependent on an interaction between mesoderm and ectoderm during gastrulation and can be used as a marker of neural induction. XIF1 shows 94% homology and XIF3 83% homology to hamster vimentin at the amino acid level over a region of the protein. Although XIF1 and XIF3 show more homology to vimentin than to any other intermediate filament gene, they have distinct temporal and spatial patterns of expression. XIF1 expression most resembles that of vimentin in higher vertebrates, being expressed in embryonic myotome and nerve cord, whilst XIF3 is unusual in that its expression is restricted predominantly to the head in tailbud embryos.     

Structure and evolutionary origin of the gene encoding mouse NF-M, the middle-molecular-mass neurofilament protein

We describe the complete sequence of the gene encoding mouse NF-M, the middle-molecular-mass neurofilament protein. The coding sequence is interrupted by two intervening sequences which align perfectly with the first two intervening sequences in the gene encoding NF-L (the low-molecular-mass neurofilament protein); there is no intron in the gene encoding NF-M corresponding to the third intron in NF-L. Therefore, both the number of introns and their arrangement in the genes coding NF-L and NF-M contrast sharply with the number and arrangement of introns in the genes of known sequence, encoding other members of the intermediate filament multigene family (desmin, vimentin, glial fibrillary acidic protein and the acidic and basic keratins); with the exception of a single truncated keratin gene that lacks an encoded tailpiece, these genes all contain eight introns, of which at least six are placed at homologous locations. Assuming the existence of a primordial intermediate filament gene containing most (if not all) the introns found in contemporary non-neurofilament intermediate filament genes, it seems likely that an RNA-mediated transposition event was involved in the generation of an ancestral gene encoding the NF polypeptides. A combination of insertional transposition and gene-duplication events could then explain the anomalous number and placement of introns within these genes. Consistent with this notion, we show that the genes encoding NF-M and NF-L are linked.     

Structure and expression of the Chinese hamster thymidine kinase gene.

My colleagues and I have cloned a nearly full-length Chinese hamster thymidine kinase (TK) cDNA in a lambda gt10 vector and characterized this cDNA by nucleotide sequencing. The hamster TK protein is encoded in this cDNA by a 702-base-pair open reading frame which specifies a 25,625-dalton protein closely homologous to the previously described human and chicken TK proteins. Using cDNA nucleotide sequence data in conjunction with sequence data derived from selected subclones of the hamster TK gene recombinant phage lambda HaTK.5, we have resolved the structure of the TK gene, finding the 1,219 base pairs of the cDNA sequence to be distributed through 11.2 kilobases of genomic DNA in at least seven exon segments. In addition, we have constructed a variety of Chinese hamster TK minigenes and exonuclease III-S1 derivatives of these genes which have permitted us to define the limits of the Chinese hamster TK gene promoter and demonstrate that efficient TK transformation of Ltk- cells by TK minigenes depends on the presence of both TK intervening sequences and sequences 3' to the site of mRNA polyadenylation.     

The 66-kDa neurofilament protein (NF-66): Sequence analysis and evolution

A 2.5 kb cDNA clone encoding the mouse 66 kd neurofilament protein (NF-66) was isolated and sequenced. The deduced protein sequence contains 501 amino acid residues. Comparison of the mouse, rat and human NF-66 indicated >90% homology in protein sequence and 85% homology in coding nucleotide sequence. A high degree of homology was observed between NF-66 and other intermediate filament proteins especially in the α-helical domain. Zooblot analyses suggested that the putative ancestral gene for vimentin and NF-66 was detectable in the avian. By comparison, the ancestral sequence for GFAP appeared after that for vimentin. Special issue dedicated to Dr. Marion E. Smith.     

Chicken riboflavin-binding protein. cDNA sequence and homology with milk folate-binding protein

The Rd gene is expressed in the livers and oviducts of laying hens and codes for the riboflavin-binding protein (RfBP) of egg yolk and egg white. A lambda gt11 cDNA library derived from chicken oviduct poly(A)+ RNA was screened with polyclonal rabbit antiserum to chicken RfBP. Positive clones were isolated and rescreened with a mixed oligonucleotide probe corresponding to residues 20-25 of the mature protein. The largest cDNA clone (969 base pairs) was subcloned into plasmid pIBI21, and the nucleotide sequence was determined by the dideoxynucleotide method. This clone contained the entire coding region for RfBP. The published amino acid sequence of the mature protein was confirmed. In addition, the following 17-residue signal peptide was deduced: Met-Leu-Arg-Phe-Ala-Ile-Thr-Leu-Phe-Ala-Val-Ile-Thr-Ser-Ser-Thr-Cys. Unexpectedly, the nucleotide sequence codes for 2 adjacent arginine residues at the carboxyl terminus that are not observed in the mature protein. The amino acid sequence of RfBP is homologous with bovine milk folate-binding protein. Eight of the nine pairs of cysteines involved in disulfide bonds in RfBP are conserved in folate-binding protein, as are all of the tryptophan residues. Sequence identity between homologous regions of these two vitamin-binding proteins is more than 30%.     

鸡补体分子C3d的基因克隆及结构分析

目的:克隆鸡补体分子C3d基因并解析其结构特点。方法:将已发表的人、小鼠、地鼠、奶牛、野兔、猪、猩猩、绵羊的C3d基因同鸡的C3α链进行序列比较分析,发现在鸡的C3α链上有一段约897bp的序列同上述动物有较高的同源性,在上下端保守区域设计一对引物788bp,应用RT-PCR扩增鸡C3d部分基因,并克隆到pMD18-T载体中,测序正确后再在上下端分别设计一对引物,理论长度分别为378和336bp,最后用3种PCR产物延伸扩出C3d全长序列。结果:获得了鸡C3d基因重组质粒pMD18-C3d,序列分析表明所获的鸡C3dcDNA全长为993bp,编码331个氨基酸残基。鸡与上述人或动物C3d核苷酸的同源性分别为66.6%、66.2%、67.7%、66.2%、67.1%、67.0%、59.6%、67.1%,与其编码的氨基酸的同源性分别为61.5%、61.9%、61.2%、61.9%、56.5%、61.9%、54.5%、61.5%;而哺乳动物间C3d的核苷酸和氨基酸的同源性则分别为74.2%～100%和72.2%～100%。进化树反映出C3d基因具有种的多样性,亲缘关系越近,进化关系也越近。结论:鸡C3d与其他动物的C3d在抗原结合位点上没有氨基酸的变化,而与CR2结合的28肽区氨基酸差异明显,说明鸡C3d结合抗原没有专一性,而结合免疫细胞则有种的特异性,由此可以推测鸡C3d只能增强鸡的特异性免疫反应。     

Structure of the gene encoding peripherin, an NGF-regulated neuronal-specific type III intermediate filament protein

We have cloned the rat gene encoding peripherin, a neuronal-specific intermediate filament protein that is NGF-regulated. Determination of the complete sequence, including 821 nucleotides of the 5'-flanking region, allows us to make conclusions about the evolutionary origin of the peripherin gene, its homology with other intermediate filament proteins, and possible mechanisms of regulation of peripherin expression in neurons. The positions of the eight peripherin gene introns correspond to the intron patterns of desmin, vimentin, and GFAP, with one example of intron sliding. Together with protein sequence homologies, this conclusively demonstrates that peripherin is a type III intermediate filament protein. The peripherin promoter contains sequences homologous to regions of other NGF-regulated promoters, which may function in peripherin induction by NGF.     

The human mid-size neurofilament subunit: a repeated protein sequence and the relationship of its gene to the intermediate filament gene family.

We report the isolation and sequence of cDNA and genomic clones for one of the two large subunits of human neurofilament, NF-M. Analysis of the sequence has allowed us to investigate the structure of the carboxy-terminal tail of this protein, and to compare it to that of the small neurofilament as well as to other intermediate filaments. The carboxy-terminal region of the protein contains a 13 amino acid proline- and serine-rich sequence repeated six times in succession. Within each repeat unit are two smaller repeats of the sequence Lys-Ser-Pro-Val. The four amino acid repeat may represent a kinase recognition site in a region of the protein that is known to be highly phosphorylated. We also note the presence of an additional heptad repeat at the extreme carboxy terminus of the protein. This region of 60 amino acids may be involved in coiled-coil interactions similar to those that facilitate the filament formation in the rod region. The human gene contains only two introns. Their positions correspond to two of the three introns found in the small neurofilament of the mouse. Thus, two of the three neurofilament genes of mammals have similar structures which are quite different from those of the other intermediate filaments. This finding suggests a common origin of the neurofilament subunits, whose evolutionary relationship to other intermediate filament genes is uncertain.     

应用RACE法克隆鸽恒定链基因的研究

为比较禽类恒定链的结构和功能, 应用RACE (Rapid Amplification of cDNA Ends) 技术首次克隆并鉴定了鸽恒定链基因。首先用一对含高度保守的DNA片段的简并引物, 从鸽脾细胞RNA扩增部分恒定链片段, 接着测序并设计新引物分别从5′和3′RACE扩增延长该片段。最后根据全基因的序列设计上、下游引物,获得大小为1 050 bp的全长cDNA。比较核苷酸序列, 鸽与鸡的Ii链同源性达到82.8％, 而与人等其它动物的同源性则在52.0％以上; 其中633 bp的开放阅读框编码211个氨基酸残基的前体蛋白。推导和分析氨基酸序列表明, 分子结构与鸡恒定链相似, 其中有些氨基酸残基表现出较高的保守性。     

Sequence of cDNAs encoding actin depolymerizing factor and cofilin of embryonic chicken skeletal muscle: two functionally distinct actin-regulatory proteins exhibit high structural homology.

Two actin-regulatory proteins of 19 and 20 kDa are involved in the regulation of actin assembly in developing chicken skeletal muscle. They are homologous with actin depolymerizing factor (ADF) and cofilin, a pH-dependent actin-modulating protein, which were originally discovered in chicken and mammalian brain, respectively. In this study, full-length cDNA clones were isolated by screening a lambda gt11 cDNA library constructed from poly(A+) RNA of embryonic chicken skeletal muscle with the antibodies specific for each protein, and their complete sequences were determined. The chicken cofilin cDNA encoded a protein of 166 amino acids, the sequence of which had over 80% identity with that of porcine brain cofilin. The amino acid sequence of the ADF was 165 amino acids and showed about 70% identity with either chicken or mammalian cofilin, in spite of the fact that ADF and cofilin are functionally distinct. Like chicken and mammalian cofilin, ADF contained a sequence similar to the nuclear transport signal sequence of SV40 large T antigen. ADF and cofilin shared a hexapeptide identical with the amino-terminal sequence of tropomyosin as well as the regions homologous to other actin-regulatory proteins, including depactin, gelsolin, and profilin. The overall nucleotide sequences and Southern blot analysis of genomic DNA, however, indicated that the two proteins were derived from different genes.     

Characterization and expression pattern of the novel MIA homolog TANGO

A novel human gene, TANGO, encoding a MIA ('melanoma inhibitory activity') homologous protein was identified by a gene bank search. TANGO, together with the homologous genes MIA, OTOR (FPD, MIAL) and MIA2 define a novel gene family sharing important structural features, significant homology at both the nucleotide and protein level, and similar genomic organization. The four members share 34-45% amino acid identity and 47-59% cDNA sequence identity. TANGO encodes a mature protein of 103 amino acids in addition to a hydrophobic secretory signal sequence. Sequence homology confirms the highly conserved SH3 structure present also in MIA, OTOR and MIA2. Thus, it appears that there are a number of extracellular proteins with SH3-fold like structures. Interestingly, in situ hybridization, RT-PCR and Northern Blots revealed very broad TANGO expression patterns in contrast to the highly restricted expression patterns previously determined for the other members of the MIA gene family. The only cells lacking TANGO expression are cells belonging to the hematopoetic system. High levels of TANGO expression were observed both during embryogenesis and in adult tissues.     

Molecular cloning and characterization of an alkalophilic Bacillus sp. C125 gene homologous to Bacillus subtilis sec Y.

A 1.8 kb HindIII DNA fragment containing the secY gene of alkalophilic Bacillus sp. C125 has been cloned into plasmid pUC119 using the B. subtilis secY gene as a probe. The complete nucleotide sequence of the cloned DNA indicated that it contained one complete ORF and parts of two other ORFs. The similarity of these ORFs to the sequences of the B. subtilis proteins indicated that they were the genes for ribosomal protein L15-SecY-adenylate kinase, in that order. The gene product of the alkalophilic Bacillus sp. C125 secY homologue was composed of 431 amino acids and its M(r) value has been calculated to be 47,100. The distribution of hydrophobic amino acids in the gene product suggested that the protein was a membrane integrated protein with ten transmembrane segments. The total amino acid sequence of alkalophilic Bacillus sp. C125 secY homologue showed 69.7% homology with that of B. subtilis secY. Regions of remarkably high homology (78% identity) were present in transmembrane regions, and cytoplasmic domains (73% identity) with less homologous regions present in extracellular domains (43% identity).