Selection and Methionine Accumulation in the Fat Body Protein 2 Gene (FBP2), a Duplicate of the Drosophila Alcohol Dehydrogenase (ADH) Gene

The Drosophila fat body protein 2 gene (Fbp2) is an ancient duplication of the alcohol dehydrogenase gene (Adh) which encodes a protein that differs substantially from ADH in its methionine content. In D. melanogaster, there is one methionine in ADH, while there are 51 (20% of all amino acids) in FBP2. Methionine is involved in 46% of amino acid replacements when Fbp2 DNA sequences are compared between D. melanogaster and D. pseudoobscura. Methionine accumulation does not affect conserved residues of the ADH-ADH^r-FBP2 multigene family. The multigene family has evolved by replacement of mildly hydrophobic amino acids by methionine with no apparent reversion. Its short-term evolution was compared between two Drosophila species, while its long-term evolution was compared between two genera belonging respectively to acalyptrate and calyptrate Diptera, Drosophila and Sarcophaga. The pattern of nucleotide substitution was consistent with an independent accumulation of methionines at the Fbp2 locus in each lineage. Under a steady-state model, the rate of methionine accumulation was constant in the lineage leading to Drosophila, and was twice as fast as that in the calyptrate lineage. Substitution rates were consistent with a slight positive selective advantage for each methionine change in about one-half of amino acid sites in Drosophila. This shows that selection can potentially account for a large proportion of amino acid replacements in the molecular evolution of proteins. Received: 12 December 1994 / Accepted: 15 April 1996     

Evidence for Gene Conversion Between Tandemly Duplicated Cytoplasmic Actin Genes of Helicoverpa armigera (Lepidoptera:Noctuidae)

Tandemly duplicated actin genes have been isolated from a Helicoverpa armigera genomic library. Sequence comparisons with actin genes from other species suggest they encode cytoplasmic actins, being most closely related to the Bombyx mori A3 actin gene. The duplicated H. armigera actin genes, termed A3a and A3b, share 98.3% nucleotide sequence identity over their entire putative coding region. Analysis of the distribution of nucleotide differences shows the first 763 bp are identical between the two coding regions, with the 18 nucleotide changes occurring in the remaining 366 bp. This observation suggests a gene conversion event has taken place between the duplicated H. armigera A3a and A3b actin genes. Translation of the open-reading frames indicates the products of these genes are identical, apart from a single amino acid difference at codon 273. Polymerase chain reaction and northern blot analysis have shown both H. armigera A3a and A3b genes are expressed during pupal development and in the brain of newly eclosed adults. A region 5′ of the H. armigera A3a actin gene start codon has been identified which contains regulatory sequences commonly found in the promoter region of actin genes, including TATA, CAAT, and CArG motifs. Received: 10 January 1996 / Accepted: 12 March 1996     

The Evolution of MHC Diversity by Segmental Duplication and Transposition of Retroelements

Sequence analysis of a 237 kb genomic fragment from the central region of the MHC has revealed that the HLA-B and HLA-C genes are contained within duplicated segments peri-B (53 kb) and peri-C (48 kb), respectively, and separated by an intervening sequence (IF) of 30 kb. The peri-B and peri-C segments share at least 90% sequence homology except when interrupted by insertions/deletions including Alu, L1, an endogenous retrovirus, and pseudogenes. The sequences of peri-B, IF, and peri-C were searched for the presence of Alu elements to use as markers of evolution, chromosomal rearrangements, and polymorphism. Of 29 Alu elements, 14 were identified in peri-B, 11 in peri-C, and 4 in IF. The Alu elements in peri-B and peri-C clustered phylogenetically into two clades which were classified as ``preduplication' and ``postduplication' clades. Four Alu J elements that are shared by peri-B and peri-C and are flanked by homologous sequences in their paralogous locations, respectively, clustered into a ``preduplication' clade. By contrast, the majority of Alu elements, which are unique to either peri-B or peri-C, clustered into a postduplication clade together with the Alu consensus subfamily members ranging from platyrrhine-specific (Spqxcg) to catarrhine-specific Alu sequences (Y). The insertion of platyrrhine-specific Alu elements in postduplication locations of peri-B and peri-C implies that these two segments are the products of a duplication which occurred in primates prior to the divergence of the New World primate from the human lineage (35–44 mya). Examination of the paralogous Alu integration sites revealed that 9 of 14 postduplication Alu sequences have produced microsatellites of different length and sequence within the Alu 3′-poly A tail. The present analysis supports the hypothesis that HLA-B and HLA-C genes are products of an extended segmental duplication between 44 and 81 million years ago (mya), and that subsequent diversification of both genomic segments occurred because of the mobility and mutation of retroelements such as Alu repeats. Received: 21 May 1997 / Accepted: 9 July 1997     

Evolution of isozyme loci and their differential tissue expression

Summary The phylogeny of the creatine kinase (CK, EC 2.7.3.2) isozyme loci and their differential tissue expressions were determined for representatives of 65 families of vertebrates, with emphasis on the fishes. The transition from the single creatine kinase locus, characteristic of certain echinoderms, to the two creatine kinase loci which are orthologous to those present in all vertebrates, occurred early in the chordate line. The majority of pre-teleostean fishes possesses only these two CK loci (A and C). These loci are relatively generalized in their tissue expressions which are variable among species of primitive fishes. The third and fourth creatine kinase loci (B and D) arose separately in the ancestors of the bony fishes and appear to be the result of regional genome duplications. Concomitant with the increase in the number of isozyme loci has been an increase in the specificity of their tissue expression. In the advanced teleost fishes the four CK loci are differentially expressed in a characteristic manner. The A₂ isozyme predominates in skeletal muscle, the B₂ isozyme in eye and brain, the C₂ isozyme in stomach muscle, and the D₂ isozyme is found exclusively in testis. We propose a phylogeny of the creatine kinase genes in the lower chordates based on the time of appearance of new CK loci, the sequence in which the loci achieve a tissue restricted expression, and the immunochemical relatedness of the orthologous and paralogous gene products.     

Genetic control of hexokinase variation in insects

Hexokinase variation in insects appears to be under the control of a single locus in some species and under multiple-locus control in others. It is often difficult to distinguish the number of loci controlling hexokinase expression. Analysis of hexokinase electrophoretic patterns in six species of mosquitoes and five species of crickets, as well as a review of hexokinase variation in other insect species, is used to emphasize the importance of interspecific comparisons when making genetic inferences. Evidence is provided which adds support for multiple hexokinase loci in dipterans. Hexokinase control by multiple loci may be difficult to determine in some species because of tight linkage, disequilibrium, and/or posttranslational modification.This work was supported by NIH Grant AI 11132-09, NSF Grant DEB-7811188, and Yale Sesil Anonymous Funds.     

On the evolution of the bacterial major sigma factors

Summary The existence of internal sequence homologies between the N-terminal halves of the gram-negative bacterial major sigma factors and their C-terminal halves, which correspond to minor factors, is reported. In the case of Escherichia-Salmonella sigma-70, an apparent homology was even found between the C-terminal helix-turn-helix DNA-binding motif and the corresponding region of the peptide N half, which, however, is not directly engaged in promoter recognition. It is proposed that major sigma factors may have originated by duplication and fusion of a DNA unit related to the ancestral gene for the whole sigma family. Coevolution of major sigma structures and complex promoters is suggested.     

Studies on the evolutionary relationships between hemoglobins inChironomus pallidivittatus andC. Tentans

Summary The monomeric hemoglobins ofChironomus tentans andC. pallidivittatus have been isolated and separated into their respective components by gel chromatography on Sephadex G-75 and ion-exchange chromatography on DEAE-Sephacel. The amino acid compositions of the purified components are given. The sequence of the 30 N-terminal amino acid residues of one of the monomeric components (Hb I fromC. pallidivittatus) was determined and found to be identical in almost all of its parts with the monomeric hemoglobins ofC. thummi (CTT III and CTT IV).Antibodies against the monomeric hemoglobins Hb I and Hb IIc and the dimeric fraction were highly specific and no cross reaction between dimeric and monomeric hemoglobins could be demonstrated. The antibodies against the monomers crossreact with the monomeric hemoglobins CTT III and CTT IV ofC. thummi. Taken together with genetic data, the immunological results indicate that divergence of monomeric from dimeric forms was an early event in the evolution of the various hemoglobins inChironomus.     

Evidence for a triplicate set of glucosephosphate isomerase structural genes in hexaploid wheat

The glucosephosphate isomerase (GPI) zymogram phenotypes of 46 aneuploid derivatives of the cultivar Chinese Spring of hexaploid wheat were determined. Variation was observed among the strains in the relative level of expression of three GPI isozymes. The relationships observed between chromosomal constitution and zymogram phenotype support the hypothesis that the three GPI isozymes are dimers composed of protomers encoded by a minimum of three homoeologous structural genes located one each in the short arms of chromosomes 1A, 1B, and 1D. The relative levels of expression per dose of chromosome arm of the products of the three arms differ in a manner consistent with the presence of a two-fold greater quantity of the product of 1BS than of the product of 1AS and of 1DS, indicating that 1BS may contain duplicate GPI structural genes.     

A new host-vector system allowing selection for foreign DNA inserts in bacteriophage lambda gtWES.

An improved vector (lambda gtWES.T5-622) for EcoRI fragments has been derived from EK2 vector lambda gtWES.lambdaB' by replacing the lambda B fragment with two identical 1.1 Md fragments from the pre-early region of bacteriophage T5. The new vector has two advantages which facilitate elimination of parental-type recombinants in an in vitro recombination experiment. Firstly, the 1.1 Md insert is too small to be re-inserted into lambda gtWES in a single copy. Secondly the 1.1 Md T5 fragment carries T5 gene A3 which prevents growth of phage retaining this fragment when the Excherichia coli host carries plasmid ColIb. Thus, essentially all plaques are due to phage with donor DNA inserts and are free of T5 DNA fragments. The size usually given as the theoretical minimum size for insertion into the lambda gt series of vectors is 0.66 Md. We have shown that this size is an underestimate and that the lower limit is about 1.6 Md. A precise estimate is difficult since there is strong selection, among phage having small inserts, for those which have acquired additional genetic material by duplication of the lambda DNA.     

Non-random intrachromosomal distribution of chromatid aberrations induced by X-rays,alkylating agents and ethanol in Vicia faba

A reconstructed karyotype of Vicia faba with all chromosomes individually distinguishable was treated with triethylene melamine (TEM), cytostasan (CYT) (a new benzimidazol nitrogen mustard), mitomycin C (MI), ethanol (EA) and X-rays. The distribution within chromosomes of induced chromatid abberations was non-random for all agents. The number of segments involved in aberration clustering corresponded to the number of sites representing constitutive heterochromatin, or the regions immediately adjacent to these, as evidenced by the position of Giemsa marker bands. Which of these potential regions of aberration clustering reacted with preferential involvement in aberrations was, in part at least, dependent upon the inducing agent used. It is argued that this may be due to differences in the base composition and/or molecular conformation of heterochromatic regions. Unexpectedly, the distribution pattern of chromatid aberrations induced by mitomycin C was found to be different from those after treatment with the alkylating agents TEM and cytostasan although mitomycin C is assumed to induce aberrations via alkylation. If mitomycin C-induced aberrations are indeed due to alkylation, this indicates that different alkylating agents do not necessarily result in identical patterns of abberation clustering. The other two alkylating agents and ethanol resulted in similar patterns of preferential distribution of abberations. X-Ray induced chromatid aberrations also showed a non-random intrachromosomal distribution, but the clustering was less pronounced than after treatment with the chemical agents.