Genomic and cDNA cloning, characterization of Delonix regia trypsin inhibitor (DrTI) gene, and expression of DrTI in Escherichia coli

Degenerate primers were designed based on all possible sequences of the N-terminal and C-terminal regions of Delonix regia trypsin inhibitor (DrTI). Five hundred sixty-one bp of polymerase chain reaction (PCR) product was amplified using the above degenerate primers and genomic DNA and cDNA of Delonix regia as a template. The amplified PCR products were cloned and sequenced. DNA sequence analysis of cDNA and genomic clones of DrTI have the same nucleotide sequence in the coding region, and manifested a genomic clone without intervening sequences in the coding region. The amino acid sequence deduced from the DrTI genomic and cDNA clones agreed with that identified via amino acid sequencing analysis, except that two amino acid residues, Ser and Lys, existed between residues Lys141 and Ser142. DrTI open reading frame was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in Escherichia coli to yield a glutathione S-transferase (GST)-fusion protein with a calculated molecular mass of about 45 kDa. The recombinant DrTI (reDrTI) was derived by treating the GST-DrTI fusion protein with thrombin. Both the reDrTI and GST-DrTI fusion protein exhibited a strong identical inhibitory effect on trypsin activity.     

The c-sis gene encodes a precursor of the B chain of platelet-derived growth factor.

The relationship between platelet-derived growth factor (PDGF) and the proto-oncogene c-sis has been determined by amino acid sequence analysis of PDGF and nucleotide sequence analysis of c-sis genomic clones. The nucleotide sequences of five regions of the human c-sis gene which are homologous to sequences of the transforming region (v-sis) of simian sarcoma virus (SSV) were determined. By alignment of the c-sis and v-sis nucleotide sequences the predicted amino acid sequence of a polypeptide homologous to the putative transforming protein p28sis of SSV was deduced. Both predicted sequences use the same termination codon and additional coding sequences may lie 5' to the homologous regions. Amino acid sequence analysis of the PDGF B chain shows identity to the amino acid sequence predicted from the c-sis sequences over 109 amino acid residues. Polymorphism may exist at two amino acid residues. These results suggest that c-sis encodes a polypeptide precursor of the B chain. A partial amino acid sequence of the PDGF A chain is also described. This chain is 60% homologous to the B chain and cannot be encoded by that part of c-sis which has been sequenced but could be encoded by sequences which lie 5' to the five regions of v-sis homology in c-sis, or at a separate locus.     

Structure and activation of the human N-ras gene

The normal human N-ras gene has been cloned. In structure and sequence it closely resembles the human H-ras and K-ras genes. The three genes share regions of nucleotide homology and nucleotide divergence within coding sequences and have a common intron/exon structure, indicating that they have evolved from a similarly spliced ancestral gene. The N-ras gene of SK-N-SH neuroblastoma cells has transforming activity, while the normal N-ras gene does not, the result of a single nucleotide change substituting lysine for glutamine in position 61 of the N-ras gene product. From previous studies we conclude that amino acid substitutions in two distinct regions can activate the transforming potential of ras gene products.     

Nucleotide sequence of two rasH related-genes isolated from the yeast Saccharomyces cerevisiae.

A complete nucleotide sequence of two ras-related yeast genes (c- rassc -1 and c- rassc -2) isolated from the yeast strain Saccharomyces cerevisiae is reported. They encode predicted polypeptides of 40,000 and 41,000 daltons, respectively. The N-terminal 170 amino acids from both genes show extensive amino acid homology to other ras genes from vertebrates, whereas their C-termini have diverged. These genes should be useful in the elucidation of a normal biological function of ras-related genes in a simple system like yeast.     

A novel cDNA extension procedure. Isolation of chicken fatty acid synthase cDNA clones

We have developed a simple and versatile cDNA extension method using lambda-exonuclease-generated single-stranded DNA as a primer. This plasmid-based cDNA extension method can be used to synthesize unidirectional extensions of the existing cDNA clones or subcloned fragments of the untranslated and exon regions of genomic DNA clones. The method is simple to use and involves no addition of linkers or tailing. We have successfully used this method to isolate 4.6 kilobase pairs of chicken fatty acid synthase cDNA clones, starting from the fragment of a genomic clone coding for the untranslated region of the fatty acid synthase mRNA. About 2.8 kilobase pairs of the cDNA coding for the chicken fatty acid synthase has been sequenced. The sequence has an open reading frame coding for 945 amino acids of the fatty acid synthase. In the sequence, we have identified the enoyl reductase, NADPH binding region, a putative beta-ketoacyl reductase region, and the entire sequences of acyl carrier protein and the thioesterase domains. The arrangement of these partial activities in this sequence confirms the arrangement of these activities as determined through partial proteolytic mapping studies. The amino acid sequence of chicken fatty acid synthase deduced from cDNA sequences shows a high degree of homology with the rat fatty acid synthase sequence, suggesting that these multifunctional proteins are conserved evolutionarily.     

Class II genes of miniature swine. IV. Characterization and expression of two allelic class II DQB cDNA clones

Two cDNA clones coding for allelic miniature swine MHC class II Ag DQB chains have been isolated, characterized, and shown to be expressed after transfection into mouse fibroblasts. The two alleles differ at the nucleotide level by an overwhelming proportion of replacement substitutions, suggesting the influence of selection for polymorphism. Most of the resulting predicted amino acid replacements are in regions commonly polymorphic in mouse Ab and human DQB sequences, corresponding to the predicted Ag recognition site. Nucleotide and amino acid sequence comparisons to homologous mouse and human sequences show more similarity between swine and man than between either swine and mouse or man and mouse. This tendency is most pronounced when comparing the 3' untranslated regions. However, an examination of unique cross-species sharing of amino acid residues suggests a closer relationship between both man and miniature swine and man and mouse than between miniature swine and mouse. The simplest explanation we can envision for these findings is that the mouse DQB gene homologue (Ab) has been subject to a higher substitution rate than either swine or human DQB genes. An additional cytoplasmic exon expressed in mouse Ab gene products and in putative human DQB2 gene products is lacking in both swine and human DQB cDNA clones. Its absence suggests either that the expression of this exon in mouse Ab genes was activated after mammalian speciation or that the expression of this exon was independently inactivated in swine DQB and human DQB1 genes. Alternatively, the mouse Ab gene may be derived from the same primordial gene as human DQB2, whereas the pig DQB gene may be derived from the same primordial gene as the human DQB1 gene.     

The Drosophila ninaE gene encodes an opsin

The Drosophila ninaE gene was isolated by a multistep protocol on the basis of its homology to bovine opsin cDNA. The gene encodes the major visual pigment protein (opsin) contained in Drosophila photoreceptor cells R1-R6. The coding sequence is interrupted by four short introns. The positions of three introns are conserved with respect to positions in mammalian opsin genes. The nucleotide sequence has intermittent regions of homology to bovine opsin coding sequences. The deduced amino acid sequence reveals significant homology to vertebrate opsins; there is strong conservation of the retinal binding site and two other regions. The predicted protein secondary structure strikingly resembles that of mammalian opsins. We conclude the Drosophila and vertebrate opsin genes are derived from a common ancestor.