Sequencing Of Coding Exons Research Articles

Genomic characterization of the human DNA excision repair-controlling gene XPAC

We have characterized the human DNA excision repair gene, XPAC (xeroderma pigmentosum group A complementing). This gene of approximately 25 kb consists of six exons. The 5'-flanking region of the gene has a CAAT box, but no TATA box. The region upstream from the coding sequence of exon 1 is G + C rich (73%), and has a GC box. Transcriptional mapping analysis suggested that there is one major transcription start point ( tsp). The presence of two polyadenylation signals suggests that the two XPAC mRNAs with different 3' untranslated regions in normal human cells are due to alternative polyadenylations. The promoter activity, measured by transient expression of the cat gene with the 5' flanking regions, indicated the presence of a functional promoter.

A mutational hot spot induced by N-hydroxy-aminofluorene in dihydrofolate reductase mutants of Chinese hamster ovary cells.

Eighteen mutants deficient in dihydrofolate reductase (DHFR) activity were induced with 0.5 microM N-hydroxy-aminofluorene in four separate experiments. This carcinogen dose killed approximately 80% of the treated cells and resulted in a mutational frequency approximately 3 x 10(-6). The nature of the induced changes in each of the mutants was determined by direct sequencing following polymerase chain reaction amplification, or in one instance, by Southern blot analysis. Nearly all (15/17) of the mutations were single base changes. Consistent with the binding specificity of this chemical, all mutations were targeted to guanine bases. The predominant change was G:C-->T:A transversion which was evident in 11/15 mutants. A single dG-AF mutational hotspot was noted at a site in the DHFR coding sequence of exon 4; one-third of the induced point mutations arose at this position. These results are compared with our previous analyses of mutants induced with the related aromatic amine, N-2-acetoxy-2-acetyl-aminofluorene.

Structure and sequence of the human homeobox gene HOX7

A cosmid containing the human sequence HOX7, homologous to the murine Hox-7 gene, was isolated from a genomic library, and the positions of the coding sequences were determined by hybridization. DNA sequence analysis demonstrated two exons that code for a homeodomain-containing protein of 297 amino acids. The open reading frame is interrupted by a single intron of approximately 1.6 kb, the splice donor and acceptor sites of which conform to known consensus sequences. The human HOX7 coding sequence has a very high degree of identity with the murine Hox-7 cDNA. Within the homeobox, the two sequences share 94% identity at the DNA level, all substitutions being silent. This high level of sequence similarity is not confined to the homeodomain; overall the human and murine HOX7 gene products show 80% identity at the amino acid level. Both the 5′ and 3′ untranslated regions also show significant similarity to the murine gene, with 79 and 70% sequence identity, respectively. The sequence upstream of the coding sequence of exon 1 contains a GC-rich putative promoter region. There is no TATA box, but a CCAAT and numerous GC boxes are present. The region encompassing the promoter region, exon 1, and the 5′ region of exon 2 have a higher than expected frequency of CpG dinucleotides; numerous sites for rare-cutter restriction enzymes are present, a characteristic of HTF islands.

P-related sequences inDrosophila bifasciata: A molecular clue to the understanding of P-element evolution in the genusDrosophila

Two P-elements (bif1 and bif2) were isolated from a genomic library of Drosophila bifasciata. Both elements are internally deleted and have lost the coding capacity for a functional transposase. One of the elements (bif2) contains an insert consisting of a repetitive sequence. The terminal inverted repeats and the segments necessary for passive mobility are well conserved. Element bif2 has retained rudiments of the coding sequence of exon 0 and exon 3, but the reading frame is destroyed by insertions and deletions. The comparison of the D. bifasciata P-elements with P-elements of Drosophila melanogaster and Drosophila nebulosa reveals that the two latter sequences are more similar to each other than either of them is to the D. bifasciata elements. This finding contradicts the phylogenetic relationship of the species and can be taken as an indirect but unequivocal evidence for recent horizontal gene transfer from a relative of D. nebulosa to the gene pool of D. melanogaster. The P-elements of D. bifasciata are phylogenetically ancient and have evolved independently for about 50 million years. A higher substitution rate at the third codon position as well as a predominance of conservative replacements at the amino acid level indicates that the P-elements of D. bifasciata have been under selective constraint over a long period and that immunobilization has occurred only recently.

Structure of a human serum amyloid A gene and modulation of its expression in transfected L cells.

The structure of a human serum amyloid A (SAA) genomic clone (SAAg9) has been analyzed and the nucleotide sequence of the coding regions is compared with that of the cDNA for apoSAA1. The leader and coding sequences of exons 2 and 3 are identical to SAA1. However, there are 10 nucleotide and 7 derived amino acid substitutions in exon 4. These changes are identical to the amino acid sequence of the amyloid protein associated with familial Mediterranean fever. In particular, the amino acid substitution (Thr to Phe) at residue 69 of SAA1 may have an important role in this type of hereditary amyloidosis. The genomic clone SAAg9 has been transfected into mouse L cells, and constitutive expression of human specific mRNA and protein were observed in stable transfected clones. The expression of both SAA mRNA and protein were increased by incubation of the transfected cells with purified human interleukin-1 (IL-1), both human and mouse recombinant IL-1, and recombinant human tumor necrosis factor alpha. The induction of SAA is pretranslational and is likely to be mediated by protein factor(s) since incubation with cycloheximide diminished IL-1-dependent increase in SAA mRNA.

Structure of the mouse gene encoding CD4 and an unusual transcript in brain.

The T-cell differentiation antigen CD4 plays an important role in the function of T cells that recognize class II major histocompatibility complex proteins. Mouse CD4 (L3T4) has previously been shown to be evolutionarily related to immunoglobulin variable regions based on the predicted protein sequence from cDNA clones. The gene encoding L3T4 was found to be transcribed not only in a subset of T-lineage cells but also unexpectedly in brain, where a shorter transcript was found. In the present study the gene encoding L3T4 is shown to span 26 kilobases and to contain 10 exons. The structural organization is similar to that of other members of the immunoglobulin gene superfamily except for the striking presence of an intron in the middle of the sequence encoding the amino-terminal immunoglobulin-like homology unit. The structure of the shorter L3T4 transcript in mouse brain has been determined. This mRNA appears to be generated from a transcriptional start site within the coding sequence in exon VI. If translated, this transcript would encode a protein of 217 amino acids that lacks the usual L3T4 signal peptide and the amino-terminal 214 amino acids of the mature protein.

Nucleotide sequence analysis of the chicken gene c- mil, the progenitor of the retroviral oncogene v- mil

The nucleotide sequence of the chicken gene c- mil was determined within and around all regions homologous to the oncogene v- mil of avian retrovirus MH2. The regions of homology to the previously determined v- mil sequence, ranging in size from 28 to 177 base pairs (bp), are distributed over 14 kilobase pairs (kbp) of the chicken genome and are organized in 11 exons. All exon-intron boundaries of c- mil, except the 5′ boundary of exon 1 and the 3′ boundary of exon 11, were unambiguously defined by the identification of consensus splice donor and acceptor sites precisely at positions where homology to v- mil ceases or resumes. The homology to v- mil starts within the coding sequence of exon 1 and ends within the 3′ untranslated region of exon 11, 12 nucleotides downstream from the nonsense codon terminating the large open reading frame shared between c- mil and v- mil. The c- mil and v- mil sequences differ at only 7 out of 1153 nucleotide positions, and the predicted sequences of v- mil and c- mil proteins differ by one conservative and four nonconservative substitutions among 379 amino acid residues. Hence, the carboxy-terminal domains of the MH2 gag-mil hybrid protein and of the putative c- mil protein are very similar. However, the amino-terminal domain of the cellular protein is possibly encoded by additional 5′ c- mil sequences not present in the transduced v- mil oncogene, while that of the MH2 hybrid protein is encoded by viral gag sequences. The sequence analysis also revealed that c- mil and c- myc derived sequences are immediately adjacent on the MH2 genome carrying both the v- mil and the v- myc oncogene. Hence, transduction of c- mil into MH2 involved recombination, at the 3′ site, with either the c- myc locus or a previously transduced v- myc gene, and, at the 5′ site, with gag sequences of the transducing virus. At both sites, no significant homologies were found between the sequence elements involved in the recombination.