Forbidden synonymous substitutions in coding regions

In the evolution of highly conserved genes, a few "synonymous" substitutions at third bases that would not alter the protein sequence are forbidden or very rare, presumably as a result of functional requirements of the gene or the messenger RNA. Another 10% or 20% of codons are significantly less variable by synonymous substitution than are the majority of codons. The changes that occur at the majority of third bases are subject to codon usage restrictions. These usage restrictions control sequence similarities between very distant genes. For example, 70% of third bases are identical in calmodulin genes of man and trypanosome. Third-base similarities of distant genes for conserved proteins are mathematically predicted, on the basis of the G+C composition of third bases. These observations indicate the need for reexamination of methods used to calculate synonymous substitutions.      

Human alpha-galactosidase A: characterization of the N-linked oligosaccharides on the intracellular and secreted glycoforms overexpressed by Chinese hamster ovary cells

Human alpha-galactosidase A (alpha-Gal A) is the lysosomal glycohydrolase that cleaves the terminal alpha-galactosyl moieties of various glycoconjugates. Overexpression of the enzyme in Chinese hamster ovary (CHO) cells results in high intracellular enzyme accumulation and the selective secretion of active enzyme. Structural analysis of the N -linked oligosaccharides of the intracellular and secreted glycoforms revealed that the secreted enzyme's oligosaccharides were remarkably heterogeneous, having high mannose (63%), complex (30%), and hybrid (5%) structures. The major high mannose oligosaccharides were Man5-7GlcNAc2 species. Approximately 40% of the high mannose and 30% of the hybrid oligosaccharides had phosphate monoester groups. The complex oligosaccharides were mono-, bi- , 2,4-tri-, 2,6-tri- and tetraantennary with or without core-region fucose, many of which had incomplete outer chains. Approximately 30% of the complex oligosaccharides were mono- or disialylated. Sialic acids were mostly N -acetylneuraminic acid and occurred exclusively in alpha2, 3-linkage. In contrast, the intracellular enzyme had only small amounts of complex chains (7.7%) and had predominantly high mannose oligosaccharides (92%), mostly Man5GlcNAc2 and smaller species, of which only 3% were phosphorylated. The complex oligosaccharides were fucosylated and had the same antennary structures as the secreted enzyme. Although most had mature outer chains, none were sialylated. Thus, the overexpression of human alpha-Gal A in CHO cells resulted in different oligosaccharide structures on the secreted and intracellular glycoforms, the highly heterogeneous secreted forms presumably due to the high level expression and impaired glycosylation in the trans- Golgi network, and the predominately Man5-7GlcNAc2 cellular glycoforms resulting from carbohydrate trimming in the lysosome.      

Genetic variation among laboratory strains of the planorbid snail Biomphalaria glabrata

Isozymes of laboratory strains of Biomphalaria glabrata have been studied by starch gel electrophoresis. Methods are outlined for adaptation of this technique to the genetic study of these snails. Twenty-eight presumptive gene loci have been identified. Twelve invariant enzymes were observed. Sixteen loci displayed some polymorphism within or among the strains. These polymorphisms were generally widespread among strains from Brazil, Puerto Rico, St. Lucia, and the Dominican Republic. A high degree of intrastrain polymorphism was noted even in some presumably inbred laboratory strains. Crosses between strains were used to demonstrate the genetic basis for the patterns observed at 9 of the 16 polymorphic loci.     

Acquisition of rifabutin resistance by a rifampicin resistant mutant of Mycobacterium tuberculosis involves an unusual spectrum of mutations and elevated frequency

Background

Mutations in a small region of the rpoB gene are responsible for most rifamycin resistance in Mycobacterium tuberculosis. In this study we have sequentially generated resistant strains to first rifampicin and then rifabutin. Portions of the rpoB gene were sequenced from 131 randomly selected mutants. Second round selection resulted in a changed frequency of specific mutations.

Methods

Mycobacterium tuberculosis (strain Mtb72) rifamycin resistant mutants were selected in vitro with either rifampicin or rifabutin. One mutant R190 (rpoB S522L) selected with rifampicin had a rifampicin MIC of 32 μg/ml but remained sensitive to rifabutin (MIC<0.8 μg/ml). This mutant was subjected to a second round of selection with rifabutin.

Results

All 105 first round resistant mutants derived from the parent strain (Mtb72) screened acquired mutations within the 81 bp rpoB hotspot. When the rifampicin resistant but rifabutin sensitive S522L mutant was subjected to a second round of selection, single additional rpoB mutations were identified in 24 (92%) of 26 second round mutants studied, but 14 (54%) of these strains contained mutations outside the 81 bp hotspot (codons 144, 146, 148, 505). Additionally, spontaneous rifabutin resistant mutants were produced at >10 times the frequency by the S522L mutant than the parent strain.

Conclusion

First round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.     

Functional variation among frugivorous birds: implications for rainforest seed dispersal in a fragmented subtropical landscape

Seed dispersal plays a critical role in rainforest regeneration patterns, hence loss of avian seed dispersers in fragmented landscapes may disrupt forest regeneration dynamics. To predict whether or not a plant will be dispersed in fragmented forests, it is necessary to have information about frugivorous bird distribution and dietary composition. However, specific dietary information for frugivorous birds is often limited. In such cases, information on the seed-crushing behaviour, gape width and relative dietary dominance by fruit may be used to describe functional groups of bird species with respect to their potential to disperse similar seeds. We used this information to assess differences in the seed dispersal potential of frugivorous bird assemblages in a fragmented rainforest landscape of southeast Queensland, Australia. The relative abundance of frugivorous birds was surveyed in extensive, remnant and regrowth rainforest sites (16 replicates of each). Large-gaped birds with mixed diets and medium-gaped birds with fruit-dominated diets were usually less abundant in remnants and regrowth than in continuous forest. Small-gaped birds with mixed diets and birds with fruit as a minor dietary component were most abundant in regrowth. We recorded a similar number of seed-crushing birds and large-gaped birds with fruit-dominated diets across site types. Bird species that may have the greatest potential to disperse a large volume and wide variety of plants, including large-seeded plants, tended to be less abundant outside of extensive forests, although one species, the figbird Sphecotheres viridis, was much more abundant in these areas. The results suggest that the dispersal of certain plant taxa would be limited in this fragmented landscape, although the potential for the dispersal of large-seeded plants may remain, despite the loss of several large-gaped disperser species.     

Independent origins of allotriploidy in the fish genus Poeciliopsis

We examined mitochondrial DNA (mtDNA) sequences and allozymes to assess possible modes of origin, clonal diversity, and evolutionary age in a triploid all-female fish of the genus Poeciliopsis from the state of Sinaloa, Mexico. Analysis of multilocus allozymes revealed that the Rio Mocorito biotype (Poeciliopsis monacha-lucida-viriosa) is trihybrid, carrying haploid genomes from three sexually reproducing species, Poeciliopsis monacha, Poeciliopsis lucida, and Poeciliopsis viriosa. Composite allozyme and mtDNA genotypes identified four clones, all bearing closely related mitochondrial haplotypes originally derived from P. monacha. Apparently these trihybrids arose endemically by addition of a haploid genome from P. viriosa, a local sexual species, to an allodiploid biotype, P. monacha-lucida, also found in the Rio Mocorito. The present analysis clearly revealed that P. monacha-lucida-viriosa arose independently of the two allotriploid biotypes that live in a river to the north (Rio Fuerte). Although the origins of allotriploidy in Poeciliopsis are less constrained phylogenetically and geographically than previously thought, known triploid biotypes all had relatively recent origins, which supports the notion that most asexual lineages are evolutionarily short-lived.     

Hybridogenetic Reproduction and Maternal Ancestry of Polyploid Iberian Fish: The Tropidophoxinellus Alburnoides Complex

Iberian minnows collectively known as the Tropidophoxinellus alburnoides STEINDACHNER complex comprise diploid and polyploid forms with highly female biased sex ratios. Previous investigators suggested that all-female clonal reproduction and interspecific hybridization may occur in this complex. We examined nuclear (allozymes) and cytoplasmic genes (mtDNA) to assess the evolutionary origins, relationships, and reproductive modes of T. alburnoides from western Spain. The multi-locus allozyme data clearly revealed the hybrid nature of all polyploid forms of this fish and some diploid forms as well. Diagnostic markers identified fish from the genus Leuciscus as the paternal ancestor of hybrids in the Duero and Guadiana River Basins. Additionally, analysis of nuclear markers revealed that hybridogenetic reproduction occurs in the diploid and triploid hybrids. The hybrids fully express the paternal Leuciscus genome and then discard it during oogenesis. Hybridogenetic ova contain only maternal nuclear genes and mtDNA from a non-hybrid T. alburnoides ancestor. Apparently diploid and triploid hybrids of T. alburnoides persist as sperm parasites on males of a sexually reproducing Leuciscus host species.     

Evolution of the Sry genes

Existing DNA sequence data on the Sry gene, the mammalian sex- determining locus in the Y chromosome, were analyzed for primates, rodents, and bovids. In all three taxonomic groups, the terminal sequences evolved faster than the HMG (high mobility group) boxes, and this applies both to synonymous (Ks) and nonsynonymous (Ka) nucleotide substitutions. Similar intragenic correlation between synonymous and nonsynonymous substitution rates was not found either in other mammalian genes that contain a conservative box (Sox, Msx) or in the MADS-box genes of plants. The rate of nonsynonymous substitutions exceeds significantly that of synonymous substitutions in the terminal Sry sequences of apes. We did not find good support for the hypothesis that the high evolutionary rate of Sry would be associated with a promiscuous mating system.      

A TEST OF THE SPONTANEOUS HETEROSIS HYPOTHESIS FOR UNISEXUAL VERTEBRATES

The coupling between clonal modes of reproduction and hybridization in unisexual vertebrates has led to the hypothesis that heterosis accounts for their ecological success (the “spontaneous heterosis” hypothesis). High levels of genic heterozygosity characteristic of unisexual-hybrid vertebrates are believed to result in enhanced growth, survivorship, and fertility relative to their sexual ancestors. To test this hypothesis, we synthesized 33 new unisexual-hybrid strains of fishes in the genus Poeciliopsis (Atheriniformes: Poeciliidae). On average, the synthetic unisexuals had lower survivorship and a higher incidence of birth defects than either of the sexual ancestors or two natural strains of unisexuals. However, a subset of these synthetic unisexuals exhibited characteristics within the range of the sexual ancestors and natural unisexual strains. These results support the alternative hypothesis that the ecological success of natural unisexuals results from selection of the most fit clones from a broad spectrum of genotypes that arose via multiple hybrid events. We propose that the coupling between unisexuality and hybridization in the vertebrates exists because hybridization is a dysgenic process that can disrupt normal gametogenesis and thus lead to clonal reproduction.