The correlation between intron length and recombination in drosophila. Dynamic equilibrium between mutational and selective forces

Intron length is negatively correlated with recombination in both Drosophila melanogaster and humans. This correlation is not likely to be the result of mutational processes alone: evolutionary analysis of intron length polymorphism in D. melanogaster reveals equivalent ratios of deletion to insertion in regions of high and low recombination. The polymorphism data do reveal, however, an excess of deletions relative to insertions (i.e., a deletion bias), with an overall deletion-to-insertion events ratio of 1.35. We propose two types of selection favoring longer intron lengths. First, the natural mutational bias toward deletion must be opposed by strong selection in very short introns to maintain the minimum intron length needed for the intron splicing reaction. Second, selection will favor insertions in introns that increase recombination between mutations under the influence of selection in adjacent exons. Mutations that increase recombination, even slightly, will be selectively favored because they reduce interference among selected mutations. Interference selection acting on intron length mutations must be very weak, as indicated by frequency spectrum analysis of Drosophila intron length polymorphism, making the equilibrium for intron length sensitive to changes in the recombinational environment and population size. One consequence of this sensitivity is that the advantage of longer introns is expected to decrease inversely with the rate of recombination, thus leading to a negative correlation between intron length and recombination rate. Also in accord with this model, intron length differs between closely related Drosophila species, with the longest variant present more often in D. melanogaster than in D. simulans. We suggest that the study of the proposed dynamic model, taking into account interference among selected sites, might shed light on many aspects of the comparative biology of genome sizes including the C value paradox.     

Selection on codon usage and base composition in Drosophila americana

We have used a polymorphism dataset on introns and coding sequences of X-linked loci in Drosophila americana to estimate the strength of selection on codon usage and/or biased gene conversion (BGC), taking into account a recent population expansion detected by a maximum-likelihood method. Drosophila americana was previously thought to have a stable demographic history, so that this evidence for a recent population expansion means that previous estimates of selection need revision. There was evidence for natural selection or BGC favouring GC over AT variants in introns, which is stronger for GC-rich than GC-poor introns. By comparing introns and coding sequences, we found evidence for selection on codon usage bias, which is much stronger than the forces acting on GC versus AT basepairs in introns.     

Evolution of the fibronectin gene. Exon structure of cell attachment domain

Genomic DNA coding for human fibronectin was identified from a human genomic library by screening with a cDNA clone that specifies the cell attachment domain in human fibronectin. Two clones which together provided more than 22 kilobase pairs of the fibronectin gene were isolated. The exons in this region correspond to approximately 40% of the coding region in the fibronectin gene. They code for the middle region of the polypeptide which consists of homologous repeating segments of about 90 amino acids called type III homologies. Nucleotide sequence of the portion of the gene corresponding to the cell attachment domain showed that the Arg-Gly-Asp-Ser cell attachment site is encoded within a 165-base pair exon. This exon, together with a 117-base pair exon codes for a homology unit. Analysis of the exon/intron organization in some of the neighboring homology units indicated a similar 2-exon structure. An exception to this pattern is that a single large exon codes for a type III homology unit that, due to alternative mRNA splicing, exists in some but not all fibronectin polypeptides. The introns separating the coding sequences for the type III homology units are located in conserved positions whereas the introns that interrupt the coding sequence within the units are in a variable position generating variations in the size of the homologous exons. This exon/intron organization suggests that the type III homology region of the fibronectin gene has evolved by a series of gene duplications of a primordial gene consisting of two exons. Specification of one of these homology units to the cell attachment domain has occurred within this exon/intron arrangement.     

Molecular variation of the shuttle craft and Lim3 genes, controlling the development of the nervous system, in a natural Drosophila melanogaster population

Comparative polymorphism of the first exon and first intron of the shuttle craft (stc) and Lim3 genes and their putative regulatory 5'-flanking sequences was analyzed using 20 sequenced natural alleles. A comparison of the stc and Lim3 genes showed that the extent of polymorphism was similar in their introns and corresponded to the variation level characteristic of Drosophila melanogaster, while the putative regulatory region and first intron of the stc gene proved to be more variable than the corresponding regions of the Lim3 gene. Since the genes under study occurred on the same chromosomes isolated from one population and were close together in a region having a high recombination rate, the difference in the extent of polymorphism between the regulatory and coding regions was explained by individual characteristics of each gene. The results made it possible to assume that the extent of polymorphism of the coding gene regions is maintained by balancing selection.     

EGFR Intron Recombination in Human Gliomas: Inappropriate Diversion of V(D)J Recombination?

The epidermal growth factor receptor (EGFR) is a membrane-bound, 170 kDa, protein tyrosine kinase that plays an important role in tumorigenesis. The EGFR gene, which is composed of over 168 kb of sequence, including a 123-kb first intron, is frequently amplified and rearranged in malignant gliomas leading to the expression of oncogenic deletion (DM) and tandem duplication (TDM) mutants. The most common DM in gliomas is EGFRvIII, which arises from recombination between introns 1 and 7 with deletion of exons 2 through 7 and intervening introns. In addition, some human gliomas express 180- to 190-kDa TDM, which are constitutively active and highly oncogenic. Both DM and TDM arise by recombination of introns that contain sequences with homology to the recombination signal sequence (RSS) heptamers and nonamers present in the V(D)J region of the immunoglobin and T lymphocyte antigen receptor genes. V(D)J RSS have also been identified in certain proto-oncogenes like bcl-2 that are involved in translocations associated with the development of human lymphomas and in other genes such as hypoxanthine-guainine phosphoribosyl transferase (HPRT) in which deletion mutations and intron rearrangements are a common phenomenon. Together with the expression of recombination associated gene (RAG) and nonhomologous end-joining (NHEJ) proteins in gliomas, these observation suggest that aberrant activity of the V(D)J recombinase may be involved in the activation of proto-oncogenes in both liquid and solid tumors.     

Protein splicing: selfish genes invade cellular proteins

Protein splicing is a series of enzymatic events involving intramolecular protein breakage, rejoining and intron homing, in which introns are able to promote the recombinative transposition of their own coding sequences. Eukaryotic and prokaryotic spliced proteins have conserved similar gene structure, but little amino acid identity. The genes coding for these spliced proteins contain internal in-frame introns that encode polypeptides that apparently self-excise from the resulting host protein sequences. Excision of the ‘protein intron’ is coupled with joining of the two flanking protein regions encoded by exons of the host gene. Some introns of this type encode DNA endonucleases, related to Group I RNA intron gene products, that stimulate gene conversion and self-transmission.     

Complex selection on intron size in Cryptococcus neoformans

We conducted a genome-wide analysis of the roles of mutation and selection in sculpting intron size in the fungal pathogen Cryptococcus neoformans. We find that deletion rate is positively associated with intron length and that insertion rate exhibits a weak negative association with intron length. These patterns suggest that long introns as well as extremely short introns in this unusually intron-rich fungal genome are in mutation-selection disequilibrium and that the proportion of constrained functional sequence in introns does not scale linearly with size. We find that untranslated region introns are longer than coding-region introns and that first introns are substantially longer than subsequent introns, suggesting heterogeneous distribution of constrained functional sequence and/or selective pressures on intron size within genes. In contrast to Drosophila, we find a positive correlation between d(N) and first intron or last intron length and a negative correlation between d(N) and internal intron length. This contrasting pattern may indicate that terminal introns and internal introns are differentially subject to hypothesized selection pressures modulating intron size and provides further evidence of widespread selective constraints on noncoding sequences.     

The Euglena gracilis chloroplast ribulose-1,5-bisphosphate carboxylase gene. I. Complete DNA sequence and analysis of the nine intervening sequences

The nucleotide sequence of 6225 base pairs (bp) of Euglena gracilis chloroplast DNA including the complete DNA sequence of the chloroplast-encoded ribulose-1,5-bisphosphate carboxylase large subunit gene along with the flanking DNA sequences is presented. The gene is greater than 5.5 kilobase pairs in length and is organized as 10 exons coding for 475 amino acids, separated by 9 introns. The exons range in size from 45 to 438 bp, while the introns range in size from 382 to 568 bp. The introns have highly conserved boundary sequences with the consensus, 5'-N GTGTGGATTT...(intron)...TTAATTTTAT N-3'. The introns are 82-85 mol% AT, with a pronounced T greater than A greater than G greater than C base bias in the RNA-like strand. They do not appear to encode any polypeptides. In addition, the introns have a conserved sequence 30-50 bp from their 3'-ends with the consensus, 5'-TACAGTTTGAAAATGA-3'. The 5'-TACA sequence bears some homology to the 5'-end of the TACTAACA sequence found in a similar location in yeast nuclear mRNA introns. The conserved sequences of the Euglena rbcL introns may be indicative of a splicing mechanism similar to that of eucaryotic nuclear mRNA introns and group II mitochondrial introns.     

The intron boundaries and flanking rRNA coding sequences of Calliphora erythrocephala rDNA

We have sequenced the available cloned examples of the intron-coding sequence junctions for the rDNA of the higher Dipteran, Calliphora erythrocephala. The introns interrupt the rDNA at the same position as the type 1 intron family detected in Drosophila melanogaster and D. virilis (10,11). A duplication of 14 base pairs of the 28S rRNA coding sequence surrounds a short version of the major genomic length class of introns. This same duplication is associated with boundaries of the type 1 introns in D. virilis and D. melanogaster (10, 13,14). We have detected considerable homology between the 3' intron sequences of C. erythrocephala and D. virilis. The rRNA coding sequences flanking the introns are extremely homologous in C. erythrocephala, D. melanogaster and D. virilis, with only one small region of significant divergence. This corresponds to a variable stem region previously identified in eukaryotic 28S rRNA at a site analogous to the L1 ribosomal protein binding site of prokaryotic 23S rRNA (27).     

Nucleotide sequence of the gene for human prothrombin

A human genomic DNA library was screened for the gene coding for human prothrombin with a cDNA coding for the human protein. Eighty-one positive lambda phage were identified, and three were chosen for further characterization. These three phage hybridized with 5' and/or 3' probes prepared from the prothrombin cDNA. The complete DNA sequence of 21 kilobases of the human prothrombin gene was determined and included a 4.9-kilobase region that was previously sequenced. The gene for human prothrombin contains 14 exons separated by 13 intervening sequences. The exons range in size from 25 to 315 base pairs, while the introns range from 84 to 9447 base pairs. Ninety percent of the gene is composed of intervening sequence. All the intron splice junctions are consistent with sequences found in other eukaryotic genes, except for the presence of GC rather than GT on the 5' end of intervening sequence L. Thirty copies of Alu repetitive DNA and two copies of partial KpnI repeats were identified in clusters within several of the intervening sequences, and these repeats represent 40% of the DNA sequence of the gene. The size, distribution, and sequence homology of the introns within the gene were then compared to those of the genes for the other vitamin K dependent proteins and several other serine proteases.