Determination of the exon structure of the distal portion of the dystrophin gene by vectorette PCR

The structure of the 3' one-third of the dystrophin gene has not previously been established. We have used vectorette PCR on a yeast artificial chromosome containing part of the human dystrophin gene to determine that there are 20 exons in this region and to characterize adjacent intron sequences of each one. Combined with previous information on the remainder of the gene, this study shows that the coding sequence is distributed between 79 exons. We have used PCR between exons to measure the distances that separate the more closely clustered exons. Vectorette PCR products were used as probes on Southern blots to assign all the 3' exons to genomic HindIII fragments that are commonly detected in the analysis of dystrophin gene deletions. The results will be useful for determining the effect of genomic deletions on the translational reading frame, for setting up genomic PCR assays to confirm point mutations, for analyzing splice site mutations, and for investigating potential cis-acting elements involved in tissue-specific alternative splicing. Vectorette PCR using primers derived from cDNA sequence represents an efficient and widely applicable method for establishing gene structure and obtaining intron sequence flanking exons, starting from a genomic clone and a cDNA sequence.     

Structural organization of the 3'' half of the rat thyroglobulin gene

We report the structural organization of an 80 Kb segment of rat DNA, which encodes for about 40% of Thyroglobulin mRNA at the 3' end. The codogenic information included in this segment is splitted in 17 exons of homogeneous size (about 200 bp). The seven exons at the extreme 3' end have been precisely defined by DNA sequence analysis. No clear sequence homology is found among the exons, even though their coding capacity is quite similar, from 55 to 63 aminoacids residues. We located 2 hormonogenic (T4 forming) sites on the extreme 3' end of the gene in different exons. The DNA sequence coding for these functional sites shows a 70% homology in a 50 nucleotides segment. In addition we found a remnant of this sequence in other exons of the gene. Two large introns have been found on the 3' end of the gene: one is 17 Kb and the other one is more than 30 Kb long. On the basis of these findings and of preliminary studies on the remaining 5' end of the gene, we can predict that the minimum length of the rat TGB gene will be 150 Kb, which makes this gene the largest so far identified eukaryotic gene. We propose in addition that the 3' end exons arose by duplication of a common ancestor.     

Computational and experimental analyses reveal previously undetected coding exons of the KRIT1 (CCM1) gene

A notable difficulty in annotating genomic sequence is identifying the correct start codon in a gene. An important such case has been found with KRIT1, the cerebral cavernous malformation type 1 (CCM1) gene. Analysis of human and mouse genomic sequence encompassing the region containing KRIT1/Krit1 using exon/gene-prediction and comparative alignment programs revealed putative exons upstream of the previously described first exon. These additional candidate exons show significant matches to mouse and human ESTs that are contiguous with and extend upstream from the previously designated 5' end of the KRIT1 cDNA sequence. RT-PCR and 5'RACE experiments confirm the presence of four additional upstream coding exons that encode an additional 207 amino acids. Importantly, a novel frameshift mutation in one of these newly identified KRIT1 exons has been found in a CCM1 family. These data establish the authentic KRIT1 amino acid sequence and suggest that the additional KRIT1 exons may harbor mutations in other CCM1 families. In addition, these results provide another example of the utility of rigorous computational and comparative sequence analysis for refining gene structure.     

Structure of the human genomic region homologous to the bovine prochymosin-encoding gene

Two human genomic libraries were probed with bovine prochymosin (bPC) cDNA. Recombinant clones covering a genomic region homologous to the entire coding region and flanking sequences of the bPC gene were isolated. Human sequences homologous to exons of the bPC gene are distributed in a DNA fragment of 10 kb. Alignment of the human sequences and the exons of bPC reveals that the human 'exons' 1-3, 5 and 7-9 have sizes identical to the corresponding bovine exons, but a nucleotide (nt) has been deleted in the human exon 4 and two nt in the human exon 6. The aligned human sequence and the coding part of bPC gene share 82% nt homology, the value ranging, in separate exons, from 76 (exon 1) to 84% (exons 5 and 6). 150 bp of 5'-flanking sequence of the human gene has 75% homology to the corresponding region of bPC gene and contains a TATA-box in a similar position. A 1-nt deletion in the human exon 4 would shift the translational reading frame of a putative human PC mRNA relative to bPC mRNA, and result in an in-phase terminator spanning codons 163 and 164 in bPC mRNA. Another terminator in-phase with the amino-acid sequence encoded by the bPC gene occurs in the human exon 5 and the second frameshift mutation in exon 6. Thus, the nt sequence analysis of the human genomic region has revealed the presence of mutations that have rendered it unable to produce a full-length protein homologous to bPC and, therefore, we refer to this gene as a human prochymosin pseudogene (hPC psi). Blot-hybridization analysis of human genomic DNA indicates that hPC psi is a single gene in the human genome.     

Origin of structural domains of the serum-albumin gene family and a predicted structure of the gene for vitamin D-binding protein

We have recently determined complete DNA sequences for the human albumin and alpha-fetoprotein [AFP] genes and thus have identified their detailed structures. Each is composed of three domains of four exons, three of which are internal and one of which is a domain-linking exon. Equivalent exons in each domain show sufficient sequence and structural similarity to be considered homologous; additional unique exons at each end of the gene show no similarity to the internal triplicated structures. Since earlier, conflicting evolutionary models were based on analysis of single gene structures, we derived from five genes a series of consensus sequences representing the three internal exons as well as the domain-linking exon. The five genes were human and rat albumin and human, mouse, and rat AFP genes. Structurally equivalent exons of the different domains are shown to have arisen from a single exon in a one-domain precursor. Exons that bridge the domains arose from an unequal crossover that fused two exons of the precursor. Our model suggests that part of the coding sequence of the one-domain precursor may have been derived from an intron, by way of loss of a splice site. The consensus sequences were used to propose an intron-exon structure for the related gene encoding the serum vitamin D-binding protein (DBP). DBP is truncated relative to albumin and AFP, and we submit that this results from deletion of two internal exons in the third domain of the gene rather than from premature termination of the coding sequence.     

The organization of the prosystemin gene

The organization of the gene encoding tomato prosystemin, a 200 amino acid protein precursor of the 18 amino acid polypeptide inducer of proteinase inhibitor synthesis in tomato and potato plants, is reported. The prosystemin sequence reveals that the gene, which is composed of five homologous pairs of exons plus a non-homologous exon at the C-terminus containing the systemin sequence, has evolved by several gene duplication-elongation events from a much smaller ancestral gene. The nucleotide and amino acid sequence homologies among the exons suggest that a small ancestral gene was duplicated to form at least two tandem repeats, followed by subsequent duplication-elongation events that resulted in five tandemly repeated nucleotide sequences and three duplicated amino acid sequence elements. Since the systemin nucleotide or amino acid sequence was not duplicated, it was either not part of the gene duplication-elongation events or its coding region evolved separately and may even have been added to the tandemly repeated part of the gene at a later time.     

Structural organization of the human kininogen gene and a model for its evolution

The entire human kininogen gene has been isolated as a set of overlapping genomic DNA fragments, and the 11 exons encompassing approximately 27 kilobase pairs have been mapped by restriction enzyme analysis and nucleotide sequence determination. The nine 5'-terminal exons encode the 5'-untranslated region and the protein-coding region for the signal peptide and the heavy chain, which are common for high molecular weight (HMW) and low molecular weight (LMW) prekininogen mRNAs. Exon 10 consists of the common sequence for bradykinin and the immediately following unique sequence for HMW prekininogen mRNA. Exon 11 is then located following a 90-nucleotide sequence downstream from exon 10 and precisely specifies the sequence unique to LMW prekininogen mRNA. This, together with the hybridization analysis of total human cellular DNA, leads us to conclude that human HMW and LMW prekininogen mRNAs are produced from a single gene as a consequence of alternative RNA processing events. The structural analysis of the kininogen gene also shows that each of the nine 5'-terminal exons discretely specifies the nine protein domains observed in the amino-terminal portion of the kininogens. Furthermore, these nine genetic domains can be characterized by a thrice repeated pattern of three genetic segments, and two sets of these three domains, encompassing exons 3-5 and exons 6-8, are most closely related to each other. Therefore, we have proposed two successive duplication mechanisms as a model for the generation of the structure of the kininogen gene.     

Structure of the human type I DNA topoisomerase gene

We describe the molecular organization of the human gene coding for type I DNA topoisomerase. The coding sequence is split into 21 exons distributed over at least 85 kilobase pairs (kb) of human genomic DNA. The sizes of the 20 introns vary widely between 0.2 and at least 30 kb and all contain the sequence elements known to be required for pre-mRNA splicing. Several of the intron sequences separate exons encoding parts of the enzyme that are highly conserved between human and yeast suggesting that at least some of the exons may code for individual, structurally, or functionally important domains of the enzyme. We also describe the promoter sequence of the human topoisomerase I gene and show that it is composed of distinct functional elements.     

Structure and analysis of the bovine atrial natriuretic peptide precursor gene

The isolation and sequence analysis of the gene encoding the bovine atrial natriuretic peptide (ANP) precursor is described. The bovine-ANP coding sequences are located on three exons which are interrupted by two intervening sequences. Comparison of the bovine, human, rat and mouse ANP gene sequences reveals a common organization of introns and exons and a high degree of sequence homology in the 5'-flanking and coding regions. Examination of the pre-proANP amino acid sequence derived from the bovine gene with those from rat, mouse and human, indicates a high degree of sequence homology in both the amino-terminal and biologically-active carboxy-terminal ANP region. The latter region in the bovine sequence resembles its human counterpart except for a carboxy-terminal Arg-Arg dipeptide.     

Elements of the rat tropoelastin gene associated with alternative splicing

Multiple isoforms of tropoelastin, the soluble precursor of elastin, are the products of translation of splice-variant mRNAs derived from the single-copy tropoelastin gene. Previous data had demonstrated DNA sequence heterogeneity in three domains of rat tropoelastin mRNA, indicating alternative splicing of several exons of the rat tropoelastin gene. Rat tropoelastin genomic clones encompassing the sites of alternative splicing were isolated and sequenced. Two sites of alternative splicing identified in rat tropoelastin mRNA sequences corresponded to exons 13-15 and exon 33 of the rat tropoelastin gene. Furthermore, the variable inclusion of an alanine codon in exon 16 resulted from two functional acceptor sites separated by three nucleotides. DNA sequences flanking exons subject to alternative splicing were analyzed. These exons contained splicing signals that differed from consensus sequences and from splicing signals of constitutively spliced exons. Introns immediately 5' of exons 14 and 33, for example, lacked typical polypyrimidine tracts and had weak, overlapping branch point sequences. Further, a region of secondary structure encompassing the acceptor site of exon 13 may influence alternative splicing of this exon. These results demonstrate that multiple cis-acting sequence elements may contribute to alternative splicing of rat tropoelastin pre-mRNA.     

Structure and evolution of the bovine prothrombin gene

The cloned bovine prothrombin gene has been characterized by partial DNA sequence analysis, including the 5' and 3' flanking sequences and all the intron-exon junctions. The gene is approximately 15.4 x 10(3) base-pairs in length and comprises 14 exons interrupted by 13 introns. The exons coding for the prepro-leader peptide and the gamma-carboxyglutamic acid-containing region are similar in organization to the corresponding exons in the factor IX and protein C genes. This region has probably evolved as a result of recent gene duplication and exon shuffling events. The exons coding for the kringles and the serine protease region of the prothrombin gene are different in organization from the homologous regions in other genes, suggesting that introns have been inserted into these regions after the initial gene duplication events.     

Structure and evolution of the human prosaposin chromosomal gene.

The gene for prosaposin was characterized by sequence analysis of chromosomal DNA to gain insight into the evolution of this locus that encodes four highly conserved sphingolipid activator proteins or saposins. The 13 exons ranged in size from 57 to 1200 bp, while the introns were from 91 to 3812 bp in length. The regions encoding saposins A, B, and D each had three exons, while that for saposin C had only two. This sequence included the regions that encode the carboxy terminus of the signal peptide, the four mature prosaposin proteins, and the 3' untranslated region. Primer extension studies indicated that over 99% of the coding sequence was contained in these 19,985 bp. Use of PCR and reverse PCR techniques indicated that the most 5' coding approximately 140 bp contained large introns and at least two small exons. Analyses of the intronic positions in the saposin regions indicated that this gene evolved from an ancestral gene by two duplication events and at least one gene rearrangement involving a double crossover after introns had been inserted into the gene.