Gly Codon Research Articles

The genomic locus of the human hemopoietic-specific cell protein tyrosine kinase (PTK)-encoding gene ( HCK) confirms conservation of exon-intron structure among human PTKs of the src family

Protein tyrosine kinases (PTKs) are implicated in the control of cell growth by virtue of their frequent appearance as products of retroviral oncogenes, as intracellular signal transducers, and as growth factor receptors or components thereof. The knowledge of the structure and sequence of family genes encoding PTKs is still limited. To date, the complete genomic structure of human src family members is only available for the C-FGR gene (encoding p55 Fgr, PTK). Sequence analysis and characterization of the intron/exon organization of the human HCK gene, encoding a hemopoietic-specific cell PTK of the src-related family, revealed a length of over 16 kb for the seven 3′-exons. All intron/exon splice junctions agree with the GT/AG rule. In each case where a boundary occurs at a Gly codon, GGG or GGA, the triplet is split between the first and second nucleotide (nt). A total of eight complete and one partial Alu repeats were identified within the introns. The nt sequence of the genomic clones resolves existing discrepancies among two published sequences of HCK cDNAs. Human HCK, S-SRC (encoding p60 Src PTK), C-FGR and LCK (encoding p56 Lck, PTK) genes thus share very similar exon/intron structures for the conserved exons. These results provide additional evidence that the different PTKs of the src-like family most likely arose by duplication of an ancestral src-like gene.

Codon usage pattern in α2(I) chain domain of chicken type I collagen and its implications for the secondary structure of the mRNA and the synthesis pauses of the collagen

A stability map of local secondary structure of the mRNA of the triple-helical α2(I) chain domain of chicken type I collagen was obtained by plotting the free energy of the optimal secondary structure of a local segment in mRNA against the segment position along a base sequence of the mRNA. It was found that the positions of the minima of free energy in the plot coincide with the positions where synthesis pauses of the α-chain polypeptides of the corresponding sizes translated from the mRNA have been reported to occur (1). The codon usage pattern of each of the three major amino acids of the α-chain domain of the collagen, Gly, Pro and Ala, fluctuates considerably along the base sequence segments of the mRNA and a deviation of the pattern from that of the average of the whole α2(I) chain domain mRNA, particularly for Gly codons, leads to a loss of the stability of the local secondary structure of the mRNA. The results suggest that selection has operated on the codon usage to optimize the secondary structure characteristic of the mRNA of the chicken collagen α2(I) chain domain which leads to a nonuniform polypeptide elongation pattern.

EnvM genes of Salmonella typhimurium and Escherichia coli.

Conjugation and bacteriophage P1 transduction experiments in Escherichia coli showed that resistance to the antibacterial compound diazaborine is caused by an allelic form of the envM gene. The envM gene from Salmonella typhimurium was cloned and sequenced. It codes for a 27,765-dalton protein. The plasmids carrying this DNA complemented a conditionally lethal envM mutant of E. coli. Recombinant plasmids containing gene envM from a diazaborine-resistant S. typhimurium strain conferred the drug resistance phenotype to susceptible E. coli cells. A guanine-to-adenine exchange in the envM gene changing a Gly codon to a Ser codon was shown to be responsible for the resistance character. Upstream of envM a small gene coding for a 10,445-dalton protein was identified. Incubating a temperature-sensitive E. coli envM mutant at the nonpermissive temperature caused effects on the cells similar to those caused by treatment with diazaborine, i.e., inhibition of fatty acid, phospholipid, and lipopolysaccharide biosynthesis, induction of a 28,000-dalton inner membrane protein, and change in the ratio of the porins OmpC and OmpF.

Substitution of valine for glycine-558 in the congenital dysthrombin thrombin Quick II alters primary substrate specificity

Thrombin Quick II is one of two dysfunctional forms of thrombin derived from the previously described congenital dysprothrombin prothrombin Quick. Thrombin Quick II does not clot fibrinogen, hydrolyze p-nitroanilide substrates of thrombin, or bind N2-[5-(dimethylamino)naphthalene-1-sulfonyl]arginine N,N-(3-ethyl-1,5-pentanediyl)amide, a high-affinity competitive inhibitor of thrombin. To determine the structural alteration in thrombin Quick II, the reduced, carboxymethylated protein was hydrolyzed by a lysyl endopeptidase. A peptide not present in a parallel thrombin hydrolysate was identified by reverse-phase chromatography. The peptide was purified by rechromatography and subjected to Edman degradation which showed that Gly-558 of human prothrombin had been replaced by Val. This corresponds to a point mutation of the Gly codon GGC to GUC. This Gly residue, which is highly conserved in the chymotrypsin family of serine proteases, forms part of the substrate binding pocket for bulky aromatic and basic side chains in chymotrypsin and trypsin, respectively. However, in porcine elastase 1, the corresponding residue is threonine. Consistent with the identified structural alteration, thrombin Quick II incorporates [3H]diisopropyl fluorophosphate stoichiometrically and hydrolyzes the elastase substrate succinyl-Ala-Ala-Pro-Leu-p-nitroanilide with a relative kcat/KM of 0.14 when compared to thrombin. This results from a 3-fold increase in KM and a 2.5-fold decrease in kcat for thrombin Quick II when compared to thrombin acting on the same substrate. These results and those of other investigators studying mutant trypsins support the conclusion that the catalytic activity of serine proteases is very sensitive to structural alterations in the primary substrate binding pocket.

Protein encoded by the third intron of cytochrome b gene in Saccharomyces cerevisiae is an mRNA maturase : Analysis of mitochondrial mutants, RNA transcripts proteins and evolutionary relationships

We have established the nucleotide sequence of the wild-type and that of a trans-acting mutant located in the third (bi3) intron of the Saccharomyces cerevisiae mitochondrial cytochrome b gene. The intron, 1691 base-pairs long, has an open reading frame 1045 base-pairs long, in phase with the preceding exon and the mutation replaces the evolutionarily conserved Gly codon of the second consensus motif by an Asp codon and blocks the formation of mature cytochrome b mRNA. Splicing intermediates of 5300 and 3900 bases with unexcised bi3 intron and a characteristic novel polypeptide (p50), the size of which corresponds to the chimeric protein encoded by upstream exons and the bi3 intronic open reading frame (ORF), accumulate in this and other bi3 splicing-deficient mutants. We conclude that the protein encoded by the bi3 ORF is a specific mRNA maturase involved in the splicing of the cytochrome b mRNA. The open reading frame of the third intron is remarkably similar to that of the unique intron of the cytochrome b gene ( cobA) of Aspergillus nidulans. Both are located in exactly the same position and possibly derive from a recent common ancestor by a horizontal transfer. We have established the nucleotide sequence of an exonic mutant located in the B3 exon. This missense mutation changes the Phe codon 151 into a Cys codon and leads to the absence of functional cytochrome b but does not affect splicing. Finally, we have studied the splicing pathway leading to the synthesis of cytochrome b mRNA by analysing, in a comprehensive manner the 22 splicing intermediates of several mutants located in bi3.

N-Methyl- N′-nitro- N-nitrosoguanidine-induced mutation in a RecA strain of Escherichia coli

274 N-methyl- N′-nitro- N-nitrosoguanidine (MNNG)-induced forward mutationsin the lacI gene of an Escherichia coli RecA − strain were cloned and sequenced. Base substitutions accounted for 264 mutations and consisted of 261 G:C → A:T transitions (including one double mutant with two G:C → A:T transitions separated by 25 base pairs), two A:T → G:C transitions and one A:T → T:A transversion. Therefore, 263 of the 274 mutations (all the transitions) can be explained as a result of the direct mispairing of O 6-methylguanine, and O 4-methylthymine residues during DNA synthesis. The source of the transversion is not known. The remaining mutations, one 16-base pair deletion, two -1 frameshifts and 7 frameshifts at the lacI frameshift hotspot, are located in runs of identical bases or flanked by directly repeated DNA sequences and can therefore be explained by template slippage events during DNA synthesis. The observed distribution of mutations recovered is identical to that found in a RecA + background indicating little involvement of RecA function in MNNG-induced mutation. Analysis of neighbouring base sequence revealed that the G:C → A:T transition was 6 times more likely to be recovered if the mutated guanine residue was preceded by a purine rather than a pyrimidine. A most striking aspect of this distribution concerns particular residues in the core domain of the lac repressor protein. Within this domain the great majority of mutations generate nonsense codons or alter Gly codons.

Nucleotide sequence of the 26 S mRNA of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and deduced sequence of the encoded structural proteins.

A cDNA clone containing all of the 26 S mRNA coding region of the RNA genome of Venezuelan equine encephalitis (VEE) virus, virulent strain Trinidad donkey (TRD), has been constructed and sequenced. The nucleotide and deduced amino acid sequences of the 26 S RNA of VEE virus conform to the general organization of the alphavirus subgenomic mRNA. Excluding the poly(A) tail, the VEE 26 S RNA is 3913 nucleotides long with a protein coding region of 3762 nucleotides. Codon usage in the translated region is nonrandom and correlates well with that reported for Sindbis (SIN), Semliki Forest (SF), and Ross River (RR) alphaviruses. Highly conserved sequences of 19 to 22 nucleotides representing putative replicase recognition sites occur at the 26 S RNA junction region of the 42 S genomic RNA and at the 3' terminus immediately preceding the poly(A) tail. The conserved sequence at the 26 S/42 S junction region of VEE virus differs from that of other alphaviruses in that an ochre termination codon (UAA) is substituted for a GGU (Gly) codon present in the other viruses. The 5' and 3' noncoding regions (30 and 121 nucleotides, respectively) of the VEE 26 S RNA are shorter than has been reported for several other alphaviruses. The approximate transmembrane domains of the VEE E1 and E2 envelope glycoproteins have been identified. VEE E1 contains a single asparagine-linked glycosylation site, whereas E2 has three such sites, all of which are apparently glycosylated. The deduced amino acid sequence of the VEE polyprotein shows an overall homology of 44 to 46% with the precursor polyproteins of SIN, SF, and RR viruses. VEE virus capsid, E1, and E2 structural proteins show 43 to 46%, 50 to 53%, and 36 to 41% homology, respectively, with the cognate proteins of SIN, SF, and RR viruses.