Isolation and characterization of cDNA clones encoding a low molecular weight nonmuscle tropomyosin isoform

cDNA clones encoding rat fibroblast tropomyosin 4 (TM-4) were isolated and characterized. DNA sequence analysis was carried out to determine the sequence of the protein. The derived amino acid sequence revealed that rat fibroblast TM-4 was found to contain 248 amino acids. The amino acid sequence of rat fibroblast TM-4 was compared with two other low molecular weight TM isoforms, equine platelet beta-TM and a human fibroblast TM. Rat TM-4 exhibited 98% sequence identity with the equine platelet TM but only 75% identity with the human fibroblast TM isoform. The high degree of conservation between the rat and equine proteins indicates that they belong to the same isotype of TM. Comparison of the amino acid sequences of the three low molecular TM isoforms along the length of the proteins reveals regions that are strongly conserved and regions that have considerably diverged. In the regions from amino acid residues 1 to 148 and 176 to 221, amino acid substitutes are moderate. The most variant regions in the sequence are in the middle part of the proteins from amino acids 149 to 175 and at the carboxyl-terminal region of the proteins from amino acids 222 to 248. The differences in the sequence of the rat and platelet TMs compared to the human TM may define distinct functional domains among the low molecular weight TMs. In addition, expression of tropomyosin was studied in a variety of tissues and transformed cells. We also demonstrate that at least three separate genes encode tropomyosins expressed in rat fibroblasts.     

Alternative splicing of Drosophila Dscam generates axon guidance receptors that exhibit isoform-specific homophilic binding

Dscam is an immunoglobulin (Ig) superfamily protein required for the formation of neuronal connections in Drosophila. Through alternative splicing, Dscam potentially gives rise to 19,008 different extracellular domains linked to one of two alternative transmembrane segments, resulting in 38,016 isoforms. All isoforms share the same domain structure but contain variable amino acid sequences within three Ig domains in the extracellular region. We demonstrate that different isoforms exhibit different binding specificity. Each isoform binds to itself but does not bind or binds poorly to other isoforms. The amino acid sequences of all three variable Ig domains determine binding specificity. Even closely related isoforms sharing nearly identical amino acid sequences exhibit isoform-specific binding. We propose that this preferential homophilic binding specificity regulates interactions between cells and contributes to the formation of complex patterns of neuronal connections.     

水稻螟虫神经肽PBAN及其受体序列的生物信息学分析

【目的】性信息素合成激活肽(PBAN)是控制昆虫产生性信息素的激素,本文旨在分析水稻螟虫神经肽PBAN及其受体的序列。【方法】通过t Blastn同源检索从水稻螟虫基因组和转录组数据库中鉴定水稻螟虫PBAN神经肽及其受体序列,在此基础上进行序列比对及系统发生分析。【结果】发现二化螟Chilo suppressalis、三化螟Tryporyza incertulas和大螟Sesamia inferens的PBAN成熟肽序列均含有33个氨基酸残基,其C端五肽序列完全相同,3种水稻螟虫PBAN多肽相似度为54.55%~63.64%;发现二化螟PBAN受体3个异构体全长氨基酸序列(PBANR-A、PBANR-B和PBANR-C),均含有7个跨膜区域。【结论】进化树分析发现不同昆虫PBAN神经肽及其受体存在一定的保守性和多样性,并且在进化树上的位置几乎与昆虫系统发育分类一致,推测PBAN神经肽和PBAN受体在昆虫系统进化过程中可能存在协同进化现象。本研究为水稻螟虫PBAN神经肽及其受体的结构和功能分析提供基础。     

Conservation of RET proto-oncogene splicing variants and implications for RET isoform function

The RET proto-oncogene encodes a receptor tyrosine kinase required for development of the kidney and neural crest-derived cell types. Alternative splicing of the 3' exons of human RET results in three protein isoforms with distinct C-termini: RET9, RET51, and RET43. These RET isoforms show differential binding to downstream adapter molecules, suggesting they may have distinct signaling functions. We have characterized Ret 3' sequences in mouse and investigated alternative splicing of this region. We found that the organization of Ret 3' sequences is very similar to human RET. The mouse locus also has alternatively spliced C-terminal coding regions, and the sequences corresponding to RET9 and RET51 are highly conserved in both position and sequence with the human locus. Further, we compared the predicted C-terminal amino acids of RET9 and RET51 in seven vertebrate species, and found that they are well conserved. We have identified sequence encoding a putative ret43 isoform in mouse, however the predicted amino acid sequence showed low homology to human RET43. Our data suggest that RET isoforms are evolutionarily highly conserved over a broad range of species, which may indicate that each isoform has a distinct role in normal RET function.     

Purification, characterization, and cDNA cloning of opine dehydrogenases from the polychaete rockworm Marphysa sanguinea

Alanopine dehydrogenase (AlDH) and three isoforms of strombine/alanopine dehydrogenase (St/AlDH) were purified from muscle tissue of the polychaete rockworm Marphysa sanguinea. The four enzymes, which can be distinguished by the isoelectric point, are monomeric 42 kDa proteins, possess similar pH-activity profiles, and display specificity for pyruvate and NAD(H). The three isoforms of St/AlDH show equivalent Km and Vmax for glycine and L-alanine and for D-strombine and meso-alanopine. Free amino acid levels in the muscle and D-strombine accumulation in vivo during muscle activity suggest that St/AlDHs function physiologically as StDH. AlDH shows specificity for L-alanine and meso-alanopine, but not for glycine or D-strombine. The amino acid sequences of AlDH and one of the St/AlDH isoforms were determined by a combination of amino acid sequence analysis and cDNA cloning. St/AlDH cDNA consisted of 1586 bp nucleotides that encode a 399-residue protein (43,346.70 Da), and AlDH cDNA consisted of 1587 bp nucleotides that encode a 399-residue protein (43,886.68 Da). The two amino acid sequences deduced from the cDNA displayed 67% amino acid identity, with greatest similarity to that of tauropine dehydrogenase from the polychaete Arabella iricolor.     

The structure of two fast-white myosin heavy chain promoters. A comparative study

Two complete myosin heavy chain genes were isolated from chicken genomic libraries, and shown to code for fast-white isoforms. Isoform specific probes were developed from the 5' nontranslated regions of the two genes and used to identify the developmental stages at which each of the genes are expressed. One of the genes is transcribed in the embryo and the other only in the adult. The 5' flanking regions of the two genes were sequenced along with the first three exons. The 5' untranslated sequences in both genes are not contiguous, one intron is present in the adult gene while the embryonic gene contains two. The promoters of both genes contain the conserved CAAT and TATA box elements observed in other eucaryotic genes. A computer assisted comparison was performed on the two genes at the nucleotide and amino acid levels. No homology could be detected in the 5' flanking regions of the genes except in and around the CAAT and TATA elements, however, structural sequences at the 5' ends were highly conserved as well as the position of the first three introns. The amino acids in and around the ATP binding site are completely conserved between the two isoforms.     

Effect of amino acid variations in the central region of human serum amyloid A on the amyloidogenic properties

Human serum amyloid A (SAA) is a precursor protein of the amyloid fibrils that are responsible for AA amyloidosis. Of the four human SAA genotypes, SAA1 is most commonly associated with AA amyloidosis. Furthermore, SAA1 has three major isoforms (SAA1.1, 1.3, and 1.5) that differ by single amino acid variations at two sites in their 104-amino acid sequences. In the present study, we examined the effect of amino acid variations in human SAA1 isoforms on the amyloidogenic properties. All SAA1 isoforms adopted α-helix structures at 4 °C, but were unstructured at 37 °C. Heparin-induced amyloid fibril formation of SAA1 was observed at 37 °C, as evidenced by the increased thioflavin T (ThT) fluorescence and β-sheet structure formation. Despite a comparable increase in ThT fluorescence, SAA1 molecules retained their α-helix structures at 4 °C. At both temperatures, no essential differences in ThT fluorescence and secondary structures were observed among the SAA1 isoforms. However, the fibril morphologies appeared to differ; SAA1.1 formed long and curly fibrils, whereas SAA1.3 formed thin and straight fibrils. The peptides corresponding to the central regions of the SAA1 isoforms containing amino acid variations showed distinct amyloidogenicities, reflecting their direct effects on amyloid fibril formation. These findings may provide novel insights into the influence of amino acid variations in human SAA on the pathogenesis of AA amyloidosis.     

Full-length rat alpha and beta cardiac myosin heavy chain sequences. Comparisons suggest a molecular basis for functional differences

The two cardiac myosin heavy chain isoforms, alpha and beta, differ functionally, alpha Myosin exhibits higher actin-activated ATPase than does beta myosin, and hearts expressing alpha myosin exhibit increased contractility relative to hearts expressing beta myosin. To understand the molecular basis for this functional difference, we determined the complete nucleotide sequence of full-length rat alpha and beta myosin heavy chain cDNAs. This study represents the first opportunity to compare full-length fast ATPase and slow ATPase muscle myosin sequences. The alpha and beta myosin heavy chain amino acid sequences are more related to each other than to other sarcomeric myosin heavy chain sequences. Of the 1938 amino acid residues in alpha and beta myosin heavy chain, 131 are non-identical with 37 non-conservative changes. Two-thirds of these non-identical residues are clustered, and several of these clusters map to regions that have been implicated as functionally important. Some of the regions identified by the clusters of non-identical amino acid residues may affect actin binding, ATP hydrolysis and force production.     

Nucleotide sequences of three different isoforms of beta-tubulin cDNA from Chinese hamster ovary cells.

The complete cDNA sequences of two clones encoding beta-tubulin isotypes and the partial sequence of a third isoform from Chinese hamster ovary cells have been determined. The deduced amino acid sequences of the three isoforms show extensive homology to each other as well as with other alpha and beta-tubulin sequences from various species. These results provide evidence for the expression of three different isoforms of beta-tubulin in Chinese hamster ovary cells.     

Molecular characterization of profilin isoforms from tobacco (Nicotiana tabacum) pollen

Profilins are actin-binding proteins in eukaryotes which participate in the phosphoinositide pathway via binding to PIP₂. Using polyclonal rabbit sera raised against plant profilins, the occurrence of several profilin isoforms is demonstrated in two-dimensionally analyzed tobacco pollen extracts. The cDNAs coding for two novel tobacco profilin isoforms (ntPro2, ntPro3) were isolated from a pollen cDNA library by antibody screening. When the cDNA and deduced amino acid sequences of the two isoforms were compared with a previously isolated tobacco pollen profilin cl)NA (ntPro1), significant differences were noted in the non-coding regions, whereas the coding sequences, in particular the functional domains, showed little variation. The cDNAs coding for the three tobacco profilin isoforms were expressed inEscherichia coli and shown to bind comparably to different anti-profilin antisera. The high degree of similarity among the different tobacco pollen profilin isoforms points to functional equivalence. Assuming that the presence of profilin is indispensable to the control of the large amounts of actin present in pollen, the occurrence of different profilin isoforms in pollen is interpreted to represent a protective mechanism against loss of profilin functions.     

Differentially expressed isoforms of the mouse retinoic acid receptor beta generated by usage of two promoters and alternative splicing.

Using anchored PCR, three different cDNA isoforms of the mouse retinoic acid receptor beta [mRAR-beta 1, mRAR-beta 2 (formerly mRAR-beta 0) and mRAR-beta 3], generated from the same gene by differential promoter usage and alternative splicing, were isolated. These three isoforms encode RAR proteins with different N-terminal A regions and identical B - F regions. The sequence encoding the first 59 amino acids of the mRAR-beta 3 A region is identical with the entire A region of mRAR-beta 1. However, the sequence of mRAR-beta 3 region A differs from that of mRAR-beta 1 by an additional 27 C-terminal amino acids encoded in an 81 nucleotide-long putative exon which is spliced in between the exons encoding the A and B regions of mRAR-beta 1. Both mRAR-beta 1 and beta 3 cDNAs differ entirely from mRAR-beta 2 in their 5'-untranslated (5'-UTR) and A region coding sequences. This N-terminal variability, in a region which was shown to be important for cell-type specific differential target gene trans-activation by other nuclear receptors, suggests that the three mRAR-beta isoforms may be functionally distinct. The conservation of RAR-beta isoform sequences from mouse to human, as seen by cross-hybridization on Southern blots or DNA sequence analysis, as well as their differential patterns of expression in various mouse tissues, corroborates this view. Additionally, the mRNA analysis data suggest that mRAR-beta 2, whose expression predominates in RA-treated embryonal carcinoma (EC) and embryonic stem (ES) cells, may be important during early stages of development. mRAR-beta 1 and beta 3, on the other hand, which are predominantly expressed in fetal and adult brain, may play some specific role in the development of the central nervous system.     

Mass spectrometrical characterisation of mouse and rat synapsin isoforms 2a and 2b

Synapsin 2 proteins are key elements of the synaptic machinery and still hold the centre stage in neuroscience research. Although fully sequenced at the nucleic acid level in mouse and rat, structural information on amino acid sequences and post-translational modifications (PTMs) is limited. Knowledge on protein sequences and PTMs, however, is mandatory for several purposes including conformational studies and the generation of antibodies. Hippocampal proteins from rat and mouse were extracted, run on two-dimensional gel electrophoresis and multi-enzyme digestion was carried out to generate peptides for mass spectrometrical analysis [nano-LC-ESI-(CID/ETD)-MS/MS]. As much as 12 synapsin 2 proteins (6 alpha and 6 beta isoforms) in the mouse and 13 synapsin 2 proteins (6 alpha and 7 beta isoforms) were observed in the rat. Protein sequences were highly identical to nucleic acid sequences, and only few amino acid exchanges probably representing polymorphisms or sequence conflicts were detected. Mouse and rat synapsins 2a differed in three amino acids, while rat and mouse synapsins 2b differed in two amino acids. As much as 13 phosphorylation sites were determined by MS/MS data in rat and mouse hippocampus and 5 were verified by phosphatase treatment. These findings are important for interpretation of previous results and design of future studies on synapsins.     

Changes in ascorbic acid content and ascorbate peroxidase activity during the development of Acetabularia mediterranea

We cloned ras-related sequences from goldfish genomic libraries constructed as recombinants using the lambda phage. Restriction enzyme mapping of the clones obtained revealed three kinds of ras-related sequences among approximately 350,000 genomic clones. One of these clones was partially sequenced. Comparison with the nucleotide sequences of mammalian ras genes showed that the determined sequences covered the predicted amino acid coding regions and parts of the intervening regions. The predicted amino acid sequences of the cloned ras-related goldfish gene suggested that the coding region is localized separately in DNA, and that its exon-intron boundaries are exactly the same as those of corresponding mammalian genes. The nucleotide and amino acid sequences of the goldfish ras-related gene may have extensive homologies to mammalian p 21 protein. Among the three mammalian ras proteins, the predicted amino acid sequence of the sequenced ras-related goldfish clone is most closely homologous (96%) to the Kirsten ras protein. Differences in the predicted amino acid sequence were greatest in the sequence predicted from the fourth exon; fewer differences were found in the sequence from the third exon, and only slight or no differences were found in the sequence predicted for the first and second exons. The 12th and 61st amino acids from the N-terminal of the protein, which are thought to be critical positions for GTP binding and catalysis, are both conserved in the goldfish protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular structure of ilvIH and its evolutionary relationship to ilvG in Escherichia coli K12.

ilvIH of Escherichia coli K12 codes for a valine-sensitive acetohydroxy acid synthase (AHASIII). The DNA sequence of ilvIH was determined. Open reading frames and appropriate translation signals exist for two polypeptides, one containing 565 amino acids (ilvI polypeptide) and the other 160 amino acids (ilvH polypeptide). A graphic matrix analysis shows three clearcut regions of homology between ilvI and ilvG (codes for AHASII). Within these three regions of homology, 50-60% of the amino acid sequences of AHASII and AHASIII are conserved. Inspection of the region between ilvG and ilvE (the K region) revealed that it can potentially code for an 86 amino acid polypeptide. A computer analysis shows small but significant homology between the predicted amino acid sequences of the N-terminal half of the ilvH polypeptide and the putative region K polypeptide. We conclude that ilvIH and ilvG-region K evolved from a common ancestor.