Analysis of cloned mRNA sequences encoding subfragment 2 and part of subfragment 1 of alpha- and beta-myosin heavy chains of rabbit heart

Two cardiac myosin heavy chain cDNA clones, pMHC alpha 252 and pMHC beta 174, were constructed using rabbit ventricular mRNA isolated from adult thyrotoxic and normal hearts, respectively. The complete DNA sequences of the 2.2- and 1.4-kilobase inserts of pMHC beta 174 and pMHC alpha 252, respectively, were obtained. The 736 amino acids specified by pMHC beta 174 begin 439 (1.3 kilobases) residues from the heavy chain NH2 terminus and include a 400-amino acid segment of subfragment 1 and the entire subfragment 2 region. Clone pMHC alpha 252 encodes 465 amino acids encompassing all of subfragment 2 and a portion of light meromyosin. Comparison of these two clones revealed extensive sequence overlap which included 1107 nucleotides specifying a 369-amino acid segment corresponding to subfragment 2. Within this region 78 (7%) base and 32 (8.7%) amino acid mismatches were noted. These differences were clustered within discrete regions, with the subfragment 1/subfragment 2 junctional region being particularly divergent. Structural differences between pMHC alpha 252 and pMHC beta 174 indicate that these two clones represent two similar but distinct myosin heavy chain genes whose expression is responsible for ventricular myosin heavy chain isoforms alpha and beta, respectively. The derived amino acid sequences of both clones exhibit extensive homology (greater than 81%) with sequences obtained by direct analysis of adult rabbit skeletal muscle myosin heavy chain protein. The sequences corresponding to the subfragment 2 region are consistent with an alpha-helical conformation with a characteristic 7-residue periodicity in the linear distribution of nonpolar amino acids. Conversely, subfragment 1 sequences specified by pMHC beta 174 suggest a folded highly irregular structure.     

A cDNA clone encoding a glycinin A1a subunit precursor of soybean.

A cDNA clone covering the whole coding region for a glycinin subunit precursor containing the A1a acidic subunit, one of the A2 family, has been identified from a library of soybean cotyledonary cDNA clones using a mixed oligonucleotide probe. Analysis of the cDNA insert revealed that it contained 1746 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 54 nucleotides, a signal peptide region corresponding to 19 amino acids, an acidic subunit region (A1a) corresponding to 291 amino acids followed by a basic subunit region corresponding to 185 amino acids, and a 3'-terminal nontranslated region of 207 nucleotides. By comparing the predicted protein sequence of this precursor with that of the legumin A precursor of pea, it was found that glycinin A2 subunit family appeared to be more closely related to the legumin than to the A3 subunit family, and that the evolutional rearrangement of glycinin genes has occurred.     

Functional complementation of truncated human skeletal-muscle chloride channel (hClC-1) using carboxyl tail fragments

Crystal structures of bacterial CLC (voltage-gated chloride channel family) proteins suggest the arrangement of permeation pores and possible gates in the transmembrane region of eukaryotic CLC channels. For the extensive cytoplasmic tails of eukaryotic CLC family members, however, there are no equivalent structural predictions. Truncations of cytoplasmic tails in different places or point mutations result in loss of function or altered gating of several members of the CLC family, suggesting functional importance. In the present study, we show that deletion of the terminal 100 amino acids (N889X) in human ClC-1 (skeletal-muscle chloride channel) has minor consequences, whereas truncation by 110 or more amino acids (from Q879X) destroys channel function. Use of the split channel strategy, co-injecting mRNAs and expressing various complementary constructs in Xenopus oocytes, confirms the importance of the Gln879-Arg888 sequence. A split between the two CBS (cystathionine b-synthase) domains (CBS1 and CBS2) gives normal function (e.g. G721X plus its complement), whereas a partial complementation, eliminating the CBS1 domain, eliminates function. Surprisingly, function is retained even when the region Gly721-Ala862 (between CBS1 and CBS2, and including most of the CBS2 domain) is omitted from the complementation. Furthermore, even shorter peptides from the CBS2-immediate post-CBS2 region are sufficient for functional complementation. We have found that just 26 amino acids from Leu863 to Arg888 are necessary since channel function is restored by co-expressing this peptide with the otherwise inactive truncation, G721X.     

Modulation of oncogenic potential by alternative gene use in human prostate cancer

Only a small percentage of primary prostate cancers have genetic changes. In contrast, nearly 90% of clinically significant human prostate cancers seems to express high levels of the nuclear phosphoprotein pp32 by in situ hybridization. Because pp32 inhibits oncogene-mediated transformation, we investigated its paradoxical expression in cancer by comparing the sequence and function of pp32 species from paired benign prostate tissue and adjacent prostatic carcinoma from three patients. Here we demonstrate that pp32 is expressed in benign prostatic tissue, but pp32r1 and pp32r2, closely-related genes located on different chromosomes, are expressed in prostate cancer. Although pp32 is a tumor suppressor, pp32r1 and pp32r2 are tumorigenic. Alternative use of the pp32, pp32r1 and pp32r2 genes may modulate the oncogenic potential of human prostate cancer.     

大豆MYB转录因子基因GmMYB174的克隆及分子特性分析

克隆获得了大豆转录因子MYB基因GmMYB174,该基因序列全长1086 bp,编码361个氨基酸,属于MYB-related家族。生物信息学分析表明大豆GmMYB174与番茄、苜蓿、葡萄等物种同源性较高,序列分析表明包含2个保守的Trp(W)位点和1个保守基序SHAQKFF。亚细胞定位结果显示GmMYB174定位于细胞核中;组织表达量显示GmMYB174在多个组织均有表达,其中在上胚轴中表达量最高。启动子元件分析表明GmMYB174包含ABRE、MYB、MYC、LTRE、GT-1等逆境胁迫应答元件。实时荧光定量PCR分析表明Gm MYB174在干旱、盐、ABA处理下均有响应。因此,GmMYB174可能参与多种胁迫应答途径。     

The complete nucleotide sequence of a small cryptic plasmid from Lactobacillus plantarum

The complete nucleotide sequence of a cryptic plasmid isolated from a Lactobacillus plantarum strain has been determined. The plasmid, designated pC30i1, has a molecular size of 2140 bp and a GC content of 37%. The sequence contains one major open reading frame (ORF R) of 951 bp, encoding a basic polypeptide of 317 amino acids, and a molecular weight of 36,956. ORF R shows extensive sequence similarity with genes coding for replication-associated proteins in a group of gram-positive plasmids known to replicate via single-stranded intermediates (ssDNA plasmids), and a stretch of 9 amino acids in the translation of ORF R closely matches a conserved region in these proteins, as well as the active site of the phi X174 Rep protein. Sequences similar to the ssDNA plasmid origins of replication are also present in the pC30i1 sequence, strengthening the hypothesis that pC30i1 belongs to the ssDNA plasmid family. The other main feature of the pC30i1 sequence is a noncoding region consisting of 14 direct, imperfect repeats of a 17-bp sequence, which may have an incompatibility function.     

Nucleotide sequence of the J gene and surrounding untranslated regions of phage G4 DNA: Comparison with phage φX174

A 290 nucleotide long region of the bacteriophage G4 genome including the end of the overlapping genes D and E, the entire gene J and the untranslated region between genes J and F has been sequenced and compared with the same region in bacteriophage φX174. Deletions, insertions, duplications and single base changes in G4 relative to φX174 have resulted in the following changes: the loss of the φX174 overlapping gene D termination and gene J initiation codons, resulting in their separation by 32 untranslated nucleotides; the deletion of one third of the gene J coding region, so that the G4 J protein is only 24 amino acids long compared with 37 amino acids in φX174; and the establishment of a longer untranslated region between G4 genes J and F, which despite many nucleotide changes retains the ability to form a stable hairpin loop in the same place and with the same geometry as in φX174. The G4 overlapping gene E is longer than in φX174 and extends beyond gene D. Sixteen nucleotides at the end of genes D and E in φX174 are duplicated in G4 before gene J.     

Molecular cloning and DNA sequence analysis of cDNA encoding chicken homologue of the Bcl-2 oncoprotein.

We have isolated a 2228 bp cDNA clone encoding a chicken homologue of the human Bcl-2 oncoprotein by low-stringency hybridization screening of a lambda gt10 cDNA library derived from a chicken B-cell lymphoma. DNA sequence analysis of this cDNA revealed an open reading frame predicting a polypeptide of 232 amino acids and an M(r) of 25,839. The predicted protein is highly homologous to the human (73%) and mouse (70%) Bcl-2 proteins, and contains a hydrophobic stretch of amino acids within its carboxyl-end (213-229) consistent with an integral membrane protein. Areas of very high sequence homology shared by all three Bcl-2 proteins at the NH2-terminus (amino acids 1-33) and within the last 150 amino acids of these proteins suggest the presence of at least two evolutionarily conserved domains within the family of Bcl-2 proteins that may be important either for their targeting to mitochondria or their ability to block programmed cell death.     

Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes.

The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.     

Analysis of the mouse gamma-crystallin gene family: assignment of multiple cDNAs to discrete genomic sequences and characterization of a representative gene.

Blot hybridization analysis of mouse DNA with gamma-crystallin-specific cDNAs has detected the presence of a multigene family comprised of at least four related genes. The detailed structure of one of these genes, mouse gamma 4-crystallin (M gamma 4.1), and its corresponding cDNA has been determined. The gene spans approximately 2.6 kilobases (kb) and contains two introns. The gene predicts a polypeptide of 174 amino acids that shares extensive sequence homology with gamma-crystallin polypeptides of other species. The two similar structural domains of the protein correspond exactly to the second and third exons of the gene, supporting an exon-duplication model of gene evolution. The similarity in structure of this gene to that recently reported for a gamma-crystallin gene of the rat (1) suggests that a common structure may exist for all gamma-crystallin genes of the two species. Moreover, a highly conserved region, 50 nucleotides in length, immediately precedes the TATA box of both the mouse and rat genes, suggesting that this sequence may be important in gene regulation.     

Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxy-terminal two-thirds of pp60c-src.

The nucleotide sequence of the 3' two-thirds of a highly conserved, molecularly cloned human cellular src gene (c-src) has been determined. This region of the c-src gene encodes the tyrosine kinase domain of the cellular src protein (pp60c-src) and corresponds to exons 6 through 12 of the chicken c-src gene, as well as nucleotides 545 to 1542 of the Rous sarcoma virus src gene (v-src). The human c-src sequence is very strongly conserved with respect to both the chicken c-src and the Rous sarcoma virus v-src genes, with nearly 90% nucleotide homology observed in this region. Amino acid sequence conservation in this region is even greater; 98% of the amino acids are conserved between human and chicken c-src. Furthermore, the exon sizes and the locations of the exon-intron boundaries are identical in the human and chicken c-src genes. However, sequences within the introns have not been conserved, and the introns within the human c-src gene are significantly larger than the corresponding introns within the chicken c-src gene. The strong amino acid conservation between the carboxy-terminal two-thirds of pp60c-src of species as divergent as humans and chickens suggests that this portion of the pp60c-src protein specifies one or more functional domains that are of great importance to some aspect of normal cellular growth or differentiation.