Chicken cardiac tropomyosin and a low-molecular-weight nonmuscle tropomyosin are related by alternative splicing

We have isolated and characterized complementary DNAs (cDNAs) encoding chicken cardiac muscle tropomyosin and a low-molecular-weight nonmuscle tropomyosin. The cardiac muscle cDNA (pCHT-4) encodes a 284-amino acid protein that differs from chicken skeletal muscle alpha- and beta-tropomyosins throughout its length. The nonmuscle cDNA (pFT-C) encodes a 248-amino acid protein that is most similar (93-94%) to the tropomyosin class including rat fibroblast TM-4, equine platelet tropomyosin, and human fibroblast TM30pl. The nucleotide sequences of the cardiac and nonmuscle cDNAs are identical from the position encoding cardiac amino acid 81 (nonmuscle amino acid 45) through cardiac amino acid 257 (nonmuscle amino acid 221). The sequences differ both 5' and 3' of this region of identity. These comparisons suggest that the chicken cardiac tropomyosin and low-molecular-weight "platelet-like" tropomyosin are derived from the same genomic locus by alternative splicing. S1 analysis suggests that this locus encodes at least one other tropomyosin isoform.     

A monoclonal antibody reactive with an activated ras protein expressing valine at position 12

Activated ras transforming genes have been described in a variety of neoplasms and encode 21,000-Dalton (p21) proteins with amino acid substitutions at positions 12, 13, and 61. In this report we describe a monoclonal antibody designated DWP that reacts specifically with synthetic dodecapeptides containing valine at position 12, to a lesser extent with peptides containing cysteine at position 12 and not with peptides containing glycine, arginine, serine, aspartic acid, glutamic acid or alanine at the same position. Western blot and immunoperoxidase studies showed that DWP specifically reacts with activated rasH or rasK proteins in NIH cells transformed by DNA from the human carcinoma cells that encode valine at position 12. DWP did not react with normal p21s encoding glycine at position 12, nor with activated p21s encoding aspartic acid, glutamic acid, arginine, serine, or cysteine at position 12. A survey of human tumor cell lines demonstrated that DWP reacted with the human bladder carcinoma cell line T24 but not with human tumor cell lines previously shown to contain other activating mutations at positions 12 or 61. DWP and perhaps additional antibodies that specifically react with alterations at positions 12 or 61 of the ras protein may be valuable in determining the presence and frequency of activated ras proteins in human malignancy.     

Activated c-N-ras in radiation-induced acute nonlymphocytic leukemia: twelfth codon aspartic acid

We describe the detection and characterization of an activated c-N-ras allele from a gamma-radiation-induced canine acute nonlymphocytic leukemia (ANLL). The activated allele was detected by use of the NIH3T3 transfection/transformation assay. The leukemia DNA had a transforming activity of 0.0125 foci/microgram. By the use of a double anti-ras antibody enzyme-linked immunoblot assay, we have dissected the lesion within the activated c-N-ras allele. Aspartic acid has been substituted for the normal glycine at position 12 in the activated p21c-N-ras. The expression of the mutant p21ras has also been detected in an in vivo passage of the radiation-induced canine ANLL from which the activated c-N-ras allele was isolated. We have demonstrated sufficient homology between canine c-N-ras genes and the human cDNA c-N-ras clone, p6a1, that allows this probe to be used in Southern blotting of canine tissues. In addition, anti-ras antibodies generated against both murine and human ras antigens are capable of detecting canine p21ras species.     

Cloning of two human liver bilirubin UDP-glucuronosyltransferase cDNAs with expression in COS-1 cells

We report the isolation and characterization of two human liver cDNA clones, HUG-Br1 and HUG-Br2; each encodes a UDP-glucuronosyltransferase enzyme which glucuronidates bilirubin IX alpha to form both the IX alpha C8 and IX alpha C12 monoconjugates and a diconjugate. HUG-Br1 cDNA (2351 base pairs) and HUG-Br2 cDNA (2368 base pairs) encode proteins with 533 and 534 amino acid residues, respectively, with a typical membrane-insertion signal peptide, membrane-spanning domain, and 3 or 5 potential asparagine-linked glycosylation sites. At the nucleic acid and deduced amino acid sequence levels the two clones are 82% similar overall, 66% similar in the amino termini, and identical after codon 287, thus encoding proteins with the same carboxyl terminus. The mRNA encoding HUG-Br1 is of high abundance, and the one encoding HUG-Br2 is of low abundance; both are 2.6 kilobases in length. Both messages (2.6 kilobases) were present in the explanted liver of a Type I Crigler-Najjar patient, although the level for that of HUG-Br1 was reduced 4.5-fold. Northern blot analysis of poly(A)+ RNA isolated from the liver of an untreated and a phenobarbital-treated Erythrocebus patas monkey with 5'-specific probes for each clone indicated that the HUG-Br2-encoded message is induced two fold, but that for HUG-Br1 is not. These data indicate that bilirubin is glucuronidated by at least two different proteins, most likely present in very different amounts. These cDNAs which encode functional bilirubin UDP-glucuronosyltransferases will allow the isolation of an appropriate gene to develop a gene therapy model for patients which have the totally deficient trait.     

cDNA cloning of ovine pulmonary SP-A, SP-B, and SP-C: isolation of two different sequences for SP-B

Pulmonary surfactant promotes alveolar stability by lowering the surface tension at the air-liquid interface in the peripheral air spaces. The three surfactant proteins SP-A, SP-B, and SP-C contribute to dynamic surface properties involved during respiration. We have cloned and sequenced the complete cDNAs for ovine SP-A and SP-C and two distinct forms of ovine SP-B cDNAs. The nucleotide sequence of ovine SP-A cDNA consists of 1,901 bp and encodes a protein of 248 amino acids. Ovine SP-C cDNA contains 809 bp, predicting a protein of 190 amino acids. Ovine SP-B is encoded by two mRNA species, which differ by a 69-bp in-frame deletion in the region coding for the active airway protein. The larger SP-B cDNA comprises 1,660 bp, encoding a putative protein of 374 amino acids. With the sequences reported, a more complete analysis of surfactant regulation and the determination of their physiological function in vivo will be enabled.     

Characterization of p21Ras-mediated apoptosis induced by protein kinase C inhibition and application to human tumor cell lines

Suppression of PKC activity can selectively induce apoptosis in cells expressing a constitutively activated p21Ras protein. We demonstrate that continued expression of p21Ras activity is required in PKC-mediated apoptosis because farnesyltransferase inhibitors abrogated the loss of viability in p21Ras-transformed cells occurring following PKC inhibition. Studies utilizing gene transfer or viral vectors demonstrate that transient expression of oncogenic p21Ras activity is sufficient for induction of apoptosis by PKC inhibition, whereas physiologic activation of p21Ras by growth factor is not sufficient to induce apoptosis. Mechanistically, the p21Ras-mediated apoptosis induced by PKC inhibition is dependent upon mitochondrial dysregulation, with a concurrent loss of mitochondrial membrane potential (psim). Cyclosporine A, which prevented the loss of psim, also inhibited HMG-induced DNA fragmentation in cells expressing an activated p21Ras. Induction of apoptosis by PKC inhibition in human tumors with oncogenic p21Ras mutations was demonstrated. Inhibition of PKC caused increased apoptosis in MIA-PaCa-2, a human pancreatic tumor line containing a mutated Ki-ras allele, when compared to HS766T, a human pancreatic tumor line with normal Ki-ras alleles. Furthermore, PKC inhibition induced apoptosis in HCT116, a human colorectal tumor line containing an oncogenic Ki-ras allele but not in a subline (Hke3) in which the mutated Ki-ras allele had been disrupted. The PKC inhibitor 1-O-hexadecyl-2-O-methyl-rac-glycerol (HMG), significantly reduced p21Ras-mediated tumor growth in vivo in a nude mouse MIA-PaCa-2 xenograft model. Collectively these studies suggest the therapeutic feasibility of targeting PKC activity in tumors expressing an activated p21Ras oncoprotein.     

Oncogenic activation of the neu-encoded receptor protein by point mutation and deletion.

The rat neu gene, which encodes a receptor-like protein homologous to the epidermal growth factor receptor, is frequently activated by a point mutation altering a valine residue to a glutamic acid residue in its predicted transmembrane domain. Additional point mutations have been constructed in a normal neu cDNA at and around amino acid position 664, the site of the naturally arising mutation. A mutation which causes a substitution of a glutamine residue for the normal valine at residue 664 leads to full oncogenic activation of the neu gene, but five other substitutions do not. Substituted glutamic acid residues at amino acid positions 663 or 665 do not activate the neu gene. Thus only a few specific residues at amino acid residue 664 can activate the oncogenic potential of the neu gene. Deletion of sequences of the transforming neu gene demonstrates that no more than 420 amino acids of the 1260 encoded by the gene are required for full transforming function. Mutagenesis of the transforming clone demonstrates a correlation between transforming activity and tyrosine kinase activity. These data indicate that the activating point mutation induces transformation through (or together with) the activities of the tyrosine kinase.     

Molecular cloning of smg p21B and identification of smg p21 purified from bovine brain and human platelets as smg p21B

We have previously purified smg p21 from bovine brain membranes and isolated its cDNA from a bovine brain cDNA library. In the present studies, we have performed extensive screening of the bovine brain cDNA library with the cloned smg p21 cDNA as a probe and isolated another cDNA encoding a protein highly homologous to smg p21. The proteins encoded by the previously and newly isolated cDNAs are designated as smg p21A and -B, respectively. Since the partial amino acid sequences determined previously from the smg p21 purified from bovine brain were identical with the common amino acid sequences between smg p21A and -B, we have further sequenced smg p21 and identified it as smg p21B. We have also further sequenced the smg p21 purified from human platelet membranes and identified it as smg p21B. Amino acid sequence analysis indicates that smg p21A is identical with the rap1A and Krev-1 proteins and smg p21B is identical with the rap1B protein.     

Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185

The neu oncogene, which is frequently activated in neuro- and glioblastomas of BDIX rats, was originally identified in the NIH 3T3 focus-forming assay. cDNA clones of the normal and transforming alleles of neu have been isolated. When these clones are inserted into the expression vector pSV2, they direct the synthesis of p185, the neu gene product. The transforming cDNA clone yields foci when transfected onto a NIH 3T3 monolayer, but the normal cDNA does not. The construction of in vitro recombinants between the normal and transforming cDNAs has allowed the determination of the mutation responsible for the activation of the neu proto-oncogene. A single point mutation changes a valine in the transmembrane domain of the predicted protein product insert to a glutamic acid. The DNAs from four independent cell lines containing activated neu oncogenes contain the identical mutation at this position.     

Deer mice: "The Drosophila of North American mammalogy"

Summary: Oocyte‐somatic cell communication is necessary for normal ovarian function. However, the identities of the majority of oocyte‐secreted proteins remain unknown. A novel cDNA encoding mouse oo cyte‐s ecreted p rotein 1 (OOSP1) was identified using a modified subtractive hybridization screen. The Oosp1 cDNA encodes a 202‐amino acid protein that contains a 21‐amino acid signal peptide sequence, 5 putative N‐linked glycosylation consensus sequences, and 6 cysteines that are predicted to form 3 disulfide bonds. OOSP1 shares amino acid identity with placental‐specific protein 1 (PLAC1), a secreted protein expressed in the placenta and the ectoplacental cone. The Oosp1 mRNA is approximately 1.0 kb and is present at high levels in the oocytes of adult ovaries and at lower levels in the spleen. The mouse Oosp1 gene is 5 exons, spans greater than 16.4 kb, and localizes to chromosome 19 at a position that shares synteny with human chromosome 11q12–11q13. The identification of OOSP1 as a new oocyte‐secreted protein permits future in vitro and in vivo functional analyses to define its role in ovarian folliculogenesis. genesis 31:105–110, 2001. © 2001 Wiley‐Liss, Inc.     

Correlation of structure with transforming activity of the P21 proteins with substitutions at positions 12 and 16

The structural effects of amino acid substitutions at positions 12 and 16 in the amino-terminal segment (Tyr 4-Ala 18) of the ras-oncogene-encoded P21 proteins have been investigated using conformational energy analysis. The P21 protein with Val at position 12 and Lys at position 16 is known to have high transforming ability, while the P21 protein with Val at position 12 and Asn at position 16 is known to have poor transforming ability, similar to that of the normal protein (with Gly at 12 and Lys at 16.) The current results demonstrate a significant conformational change at position 15 induced by the substitution of Asn for Lys at position 16, which could explain this alteration in transformation potential. These findings are consistent with previous results suggesting the existence of a normal and a malignancy-causing conformation for the P21 proteins and suggest that the critical transforming region may encompass residues 12–15.     

Sequence variation and genomic organization of fatty acid desaturase-2 (fad2) and fatty acid desaturase-6 (fad6) cDNAs in maize

Increased levels of oleic acid may enhance the nutritional and functional value of corn. Corn oil is primarily composed of palmitic, stearic, oleic, linoleic and linolenic fatty acids. Delta-12 desaturase in plants converts oleic acid (18:1) to linoleic acid (18:2) by inserting a double bond at the delta-12 position. Fatty acid desaturase-2 (fad2) encodes delta-12 desaturase that functions in the endoplasmic reticulum while fatty acid desaturase-6 (fad6) encodes delta-12 desaturase that functions in plastids. Complementary DNA (cDNA) clones from putative maize homologs for fad2 and fad6 were identified and the entire clones DNA sequenced. The maize fad2 cDNAs showed an amino-acid identity of 67-77% to fad2 of Glycine, Arabidopsis and Brassica species. Our cDNA sequence comparisons suggested that more than one fad2 gene is transcribed in maize embryos. Two different fad2 cDNAs from an embryo cDNA library map to separate chromosomal positions, providing evidence consistent with two different isoforms of fad2 expressed in the embryo. The fad2 cDNAs from multiple tissue sources clustered into three groups on a phenogram, and map to different positions on chromosomes 4, 5 and 10, which suggests at least three different isoforms of fad2 may be expressed in the maize plant. The two maize fad6 cDNAs share 81% amino-acid identity with the Arabidopsis fad6 and map to chromosome 1. Northern analysis revealed that fad2 is transcribed in embryos at 14, 21, 28 and 35 days after pollination, with the highest level observed at day 14. None of the fad2 or fad6 clones mapped to maize chromosome bins associated with QTLs for the ratio of oleic/linoleic acid, notably bin 6.04 which contains the linoleic1 locus and the largest reported QTL for the oleic/linoleic ratio. This suggests, but does not prove, that some of the QTLs for the oleic/linoleic acid ratio do not involve allelic variants of fad2 or fad6 but rather involve other genes that may influence flux through the enzymes encoded by fad2 or fad6.     

一个鼻咽癌相关EST的鉴定及其全长cDNA序列分析

鼻咽癌是我国南方及东南亚地区常见的恶性肿瘤之一.通过对鼻咽癌染色体高频率杂合性丢失区域3p21的表达序列标签(expressedsequencetag,EST)进行同源性比较分析,运用逆转录聚合酶链式反应的方法,筛选到一个在41.18%(14/34)的鼻咽癌活检组织及20.0%(1/5)的鼻咽癌细胞系中表达下调的ESTBG772301;并用Northern杂交方法,检测了该EST在多种正常成人组织中的表达状况及其所代表基因的转录本大小.在此基础上,对该EST来源的cDNA克隆(IMAGE:4839190)进行直接测序,获得了一个全长为2377bp的新cDNA序列;经生物信息学分析,发现它与已知基因序列无明显同源性,属于一个新基因,定位于染色体3p21.3,被命名为鼻咽癌表达下调基因(NPCEDRG,GenBank登录号:AF538150).其编码的蛋白质含169个氨基酸,与一个已报道的在进化上相对保守、功能未知的人类蛋白Nicolin1(简称NICN1)N端170个氨基酸残基的序列同源性为97%,但缺少NICN1蛋白C端43个氨基酸残基,可能是nicolin1基因不同剪接本的编码产物.     

Characterization of long cDNA clones from human adult spleen. II. The complete sequences of 81 cDNA clones.

To accumulate information on the coding sequences (CDSs) of unidentified genes, we have conducted a sequencing project of human long cDNA clones. Both the end sequences of approximately 10,000 cDNA clones from two size-fractionated human spleen cDNA libraries (average sizes of 4.5 kb and 5.6 kb) were determined by single-pass sequencing to select cDNAs with unidentified sequences. We herein present the entire sequences of 81 cDNA clones, most of which were selected by two approaches based on their protein-coding potentialities in silico: Fifty-eight cDNA clones were selected as those having protein-coding potentialities at the 5'-end of single-pass sequences by applying the GeneMark analysis; and 20 cDNA clones were selected as those expected to encode proteins larger than 100 amino acid residues by analysis of the human genome sequences flanked by both the end sequences of cDNAs using the GENSCAN gene prediction program. In addition to these newly identified cDNAs, three cDNA clones were isolated by colony hybridization experiments using probes corresponding to known gene sequences since these cDNAs are likely to contain considerable amounts of new information regarding the genes already annotated. The sequence data indicated that the average sizes of the inserts and corresponding CDSs of cDNA clones analyzed here were 5.0 kb and 2.0 kb (670 amino acid residues), respectively. From the results of homology and motif searches against the public databases, functional categories of the 29 predicted gene products could be assigned; 86% of these predicted gene products (25 gene products) were classified into proteins relating to cell signaling/communication, nucleic acid management, and cell structure/motility.     

Premature chromosome condensation is induced by a point mutation in the hamster RCC1 gene.

At the nonpermissive temperature, premature chromosome condensation (PCC) occurs in tsBN2 cells derived from the BHK cell line, which can be converted to the Ts+ phenotype by the human RCC1 gene. To prove that the RCC1 gene is the mutant gene in tsBN2 cells, which have RCC1 mRNA and protein of the same sizes as those of BHK cells, RCC1 cDNAs were isolated from BHK and tsBN2 cells and sequenced to search for mutations. The hamster (BHK) RCC1 cDNA encodes a protein of 421 amino acids homologous to the human RCC1 protein. In a comparison of the base sequences of BHK and BN2 RCC1 cDNAs, a single base change, cytosine to thymine (serine to phenylalanine), was found in the 256th codon of BN2 RCC1 cDNA. The same transition was verified in the RCC1 genomic DNA by the polymerase chain reaction method. BHK RCC1 cDNA, but not tsBN2 RCC1 cDNA, complemented the tsBN2 mutation, although both have the same amino acid sequence except for one amino acid at the 256th codon. This amino acid change, serine to phenylalanine, was estimated to cause a profound structural change in the RCC1 protein.