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Structural and genetic analyses of HLA class I molecules using monoclonal xenoantibodies.

The monoclonal antibodies B1.23.2 and B9.12 were developed to characterize human class I major histocompatibility gene products. They detect two different epitopes and define on a given lymphoblastoid B cell line two subsets of beta 2 microglobulin-complexed HLA class I molecules. One subset reacts with B9.12 and B1.23.2 and the other one expresses only the B9.12 epitope. Binding assays were performed on C3H mouse L cells which had been transformed with various single HLA-class I genes and the two detected molecular subsets were shown to be encoded by different genetic loci. Unlike B1.23.2, B9.12 detects all the beta 2m-complexed molecules expressed in human B cell lines and recognizes an epitope different from the one defined by W6/32. In contrast to the vast majority of the other reported anti-class I monoclonal antibodies (including B9.12), B1.23.2 recognizes an epitope expressed on both beta 2m-associated and -free HLA class I heavy chains.

Animals↗

Analysis of codon:anticodon interactions within the ribosome provides new insights into codon reading and the genetic code structure.

Although the decoding rules have been largely elucidated, the physical-chemical reasons for the "correctness" of codon:anticodon duplexes have never been clear. In this work, on the basis of the available data, we propose that the correct codon:anticodon duplexes are those whose formation and interaction with the ribosomal decoding center are not accompanied by uncompensated losses of hydrogen and ionic bonds. Other factors such as proofreading, base-base stacking and aminoacyl-tRNA concentration contribute to the efficiency and accuracy of aminoacyl-tRNA selection, and certainly these factors are important; but we suggest that analyses of hydrogen and ionic bonding alone provides a robust first-order approximation of decoding accuracy. Thus our model can simplify predictions about decoding accuracy and error. The model can be refined with data, but is already powerful enough to explain all of the available data on decoding accuracy. Here we predict which duplexes should be considered correct, which duplexes are responsible for virtually all misreading, and we suggest an evolutionary scheme that gave rise to the mixed boxes of the genetic code.

Anticodon↗

Genetic diversity and molecular identification of mosquito species in the Anopheles maculatus group using the ITS2 region of rDNA.

The species diversity and genetic structure of mosquitoes belonging to the Anopheles maculatus group in Southeast Asia were investigated using the internal transcribed spacer 2 (ITS2) of ribosomal DNA (rDNA). A molecular phylogeny indicates the presence of at least one hitherto unrecognised species. Mosquitoes of chromosomal form K from eastern Thailand have a unique ITS2 sequence that is 3.7% divergent from the next most closely related taxon (An. sawadwongporni) in the group. In the context of negligible intraspecific variation at ITS2, this suggests that chromosomal form K is most probably a distinct species. Although An. maculatus sensu stricto from northern Thailand and southern Thailand/peninsular Malaysia differ from each other in chromosomal banding pattern and vectorial capacity, no intraspecific variation was observed in the ITS2 sequences of this species over this entire geographic area despite an extensive survey. A PCR-based identification method was developed to distinguish five species of the group (An. maculatus, An. dravidicus, An. pseudowillmori, An. sawadwongporni and chromosomal form K) to assist field-based studies in northwestern Thailand. Sequences from 187 mosquitoes (mostly An. maculatus and An. sawadwongporni) revealed no intraspecific variation in specimens from Thailand, Cambodia, mainland China, Malaysia, Taiwan and Vietnam, suggesting that this identification method will be widely applicable in Southeast Asia. The lack of detectable genetic structure also suggests that populations of these species are either connected by gene flow and/or share a recent common history.

Animals↗

Dissecting genetic variance structure and evaluating genomic prediction models for single-cross hybrids derived from Stiff Stalk and Non-Stiff Stalk maize heterotic groups.

The early 20th-century discovery of heterosis and the establishment of heterotic groups transformed maize (Zea mays L.) into a keystone of global agriculture. However, maize breeding faces two significant challenges: the gradual decline of general combining ability (GCA) variance within heterotic groups and the impracticality of testing all possible single crosses in the early stages of a breeding program. Here, we developed genomic best linear unbiased prediction (GBLUP)-based multikernel models, using additive and two alternative nonadditive genomic relationship matrices, to estimate the variance components associated with the general combining ability of Stiff Stalk (SS) and Non-Stiff Stalk (NSS) heterotic groups and the specific combining ability arising from their crosses. We further applied these models to predict the performance of untested single-cross combinations under varying levels of parental information. We showed that the SS and NSS groups retained significant GCA variance across traits in both early- and late-maturity groups. The SS group, in contrast, exhibited no detectable GCA variance in grain yield for the intermediate-flowering subset of hybrids, highlighting a limitation for future genetic improvement. Furthermore, our results showed that GBLUP-based multikernel models effectively identified superior hybrids when parental information was available. In the absence of this information, however, these models underperformed compared to covariance-based approaches. Both nonadditive matrices yielded similar results, indicating that they capture comparable genetic relationship patterns despite their distinct formulations. Overall, this study sheds light on the future use of US maize commercial germplasm and demonstrates how GBLUP-based multikernel models can improve the efficiency of hybrid breeding programs.

Zea mays↗

Sequence of three bronze alleles of maize and correlation with the genetic fine structure.

The genomic sequences of three bronze alleles from Zea mays, Bz-McC, Bz-W22 and bz-R, are presented together with their flanking sequences. The bronze locus encodes UDPglucose flavonoid glucosyl-transferase (UFGT), an anthocyanin biosynthetic enzyme. The wild-type alleles Bz-McC and Bz-W22 condition purple phenotypes in the seed and plant, while bz-R conditions a bronze color. A full length cDNA corresponding to the Bz-McC allele was cloned and sequenced. Primer extension and RNase protection experiments were used to verify the 5' end of the bronze transcript. The Bz-McC allele has a 1416-bp coding region, a 100-bp intron and an approximately 83-bp 5' leader. Upstream of the message initiation site the sequences CTAACT and AATAAA occupy the positions where the eukaryotic consensus CCAAT and TATA boxes are normally found. The alleles Bz-McC and bz-R each have different large insertions with characteristics of transposable elements in their 5' flanking regions. The bz-R allele is distinguished by a 340-bp deletion starting within the intron and including 285 bp of the second exon. The Bz-McC and Bz-W22 isoalleles are known to differ in two genetically defined locations. The uts and uqv sites from the Bz-McC allele condition, respectively, lowered thermostability for the UFGT enzyme and increased amount of UFGT activity when compared with the corresponding sites in the Bz-W22 allele. The uts site maps to a region of the gene encoding two adjacent amino acid differences, either or both of which might alter the thermostability of the UFGT enzyme. The difference in UFGT levels conditioned by the uqv site is shown here to be correlated with variation in the bronze mRNA level. A likely cause of this decreased bronze mRNA level in Bz-W22 is a 6-bp duplication near the sequence CTAACT located 74 bp upstream of the bronze message initiation site. This region is therefore tentatively identified as the uqv site.

Alleles↗

Reproduction and recruitment in perennial colonies of the introduced wasp Vespula germanica.

We investigated the genetic structure of perennial colonies of the yellowjacket wasp (Vespula germanica) in its introduced range in Australia and New Zealand. The nuclear genotypes of 712 gynes from 21 colonies, 147 workers from 5 colonies, and 81 males from 4 colonies were assayed at three polymorphic microsatellite loci. The mitochondrial haplotypes of all wasps also were determined for a 450-bp region of the mtDNA using double-stranded conformational polymorphism (DSCP) analysis. We found that multiple reproductives were needed to explain the genotypes of gynes, workers, and males in 7 of 21, 2 of 5, and 2 of 4 colonies, respectively, and that nestmate relatedness of these three castes equaled 0.42, 0.16, and 0.22, respectively. The mitochondrial data revealed that all individuals shared the same mtDNA haplotype in 20 of the 21 colonies. However, in one colony, gynes and workers displayed multiple mtDNA haplotypes, indicating that nonnestmate recruitment had occurred. Overall the genetic structure within the majority of perennial colonies conformed to expectations based on the biology of V. germanica and kin selection theory for polygyne colonies; multiple reproductives successfully produced offspring and were recruited into their natal nests, thereby maintaining relatively high relatedness between interacting individuals.

Animals↗

Genetic population structure and mobility of two nectar-feeding bats from Venezuelan deserts: inferences from mitochondrial DNA.

Glossophaga longirostris and Leptonycteris curasoae are nectar-feeding bats associated with arid zones in northern South America. Despite their close phylogenetic relationship, sympatric condition and niche similarities, morphological and ecological evidence suggest that these species differ in dispersal capabilities. Using mitochondrial DNA, we tested the hypothesis that these species exhibit different levels of population structure that are congruent with their particular movement capabilities. We sequenced a section of the control region of mtDNA for 41 G. longirostris and 42 L. curasoae from 11 zones in Venezuela. Population subdivision in G. longirostris (FST = 0.725) was considerably higher than in L. curasoae (FST = 0.167). L. curasoae individuals shared haplotypes at greater distances (812 km) than G. longirostris (592 km). Our results offer preliminary evidence for one of two possible scenarios, either greater mobility in L. curasoae or a higher degree of female philopatry in G. longirostris.

Animals↗

Structural and genetic relationships between cytosolic and mitochondrial isoenzymes.

The most common type of genetic relationship between cytosolic and mitochondrial isoenzymes will probably be found to be divergent evolution from a common ancestral form. This is firmly established for the aspartate aminotransferases and less directly so in other cases. The two isoenzymes of aspartate aminotransferase have evolved at roughly equal rates at the level of total amino acid sequence but certain limited surface regions of the mitochondrial form have been much more highly conserved than corresponding regions in the cytosolic protein; these regions probably play a role in topogenesis of the mitochondrial isoenzyme. It is of interest that nearly all mitochondrial proteins are initially synthesised as precursors of molecular weight greater than the mature forms. In the case of aspartate aminotransferase, and possibly of other such isoenzymes, the N-terminus of the mature protein is nearly coincident with that of the cytosolic isoenzyme. Hence during evolution either the gene for the mitochondrial isoenzyme has gained an extra coding region for this N-terminal extension or, less likely, the structural gene for the cytosolic form has suffered a sizeable terminal deletion. Cytosolic and mitochondrial superoxide dismutases have not shared a common ancestral form as shown by the fact that their primary structures are completely unrelated. On the other hand, the mitochondrial and prokaryotic enzymes are clearly related. There is now, however, evidence to suggest that some prokaryotes possess a copper/zinc enzyme related to the eukaryotic cytosolic form. Hence the possibility arises that primitive prokaryotes possessed both proteins. The copper/zinc superoxide dismutase has been retained in the cytosol of eukaryotic cells and a few bacterial species.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Genetic population structure of coagulase-negative staphylococci associated with carriage and disease in preterm infants.

Coagulase-negative staphylococci (CoNS) are a leading cause of sepsis in the neonatal intensive care unit (NICU) setting. To evaluate the hypothesis that isolates of CoNS associated with disease belong to hypervirulent clones, as opposed to being drawn randomly from the neonatal unit carriage flora, we conducted a prospective, case-controlled study in a busy NICU. Using pulsed-field gel electrophoresis (PFGE), we compared the population structures of CoNS isolates associated with bacteremia with isolates from the skin of healthy and infected neonates and with blood culture contaminants. Endemic clones of CoNS were identified, but there was no difference in the distribution of the 6 species or 73 PFGE types between the carriage and disease isolate groups; this suggests that hypervirulent clones with an enhanced ability to cause disease were not present in this NICU setting.

Anti-Bacterial Agents↗

An experimental and theoretical study of the inhibition of Escherichia coli lac operon gene expression by antigene oligonucleotides.

Previously, we have developed a genetically structured mathematical model to describe the inhibition of Escherichia coli lac operon gene expression by antigene oligos. Our model predicted that antigene oligos targeted to the operator region of the lac operon would have a significant inhibitory effect on beta-galactosidase production. In this investigation, the E. coli lac operon gene expression in the presence of antigene oligos was studied experimentally. A 21-mer oligo, which was designed to form a triplex with the operator, was found to be able to specifically inhibit beta-galactosidase production in a dose-dependent manner. In contrast to the 21-mer triplex-forming oligonucleotide (TFO), several control oligos showed no inhibitory effect. The ineffectiveness of the various control oligos, along with the fact that the 21-mer oligo has no homology sequence with lacZYA, and no mRNA is transcribed from the operator, suggests that the 21-mer oligo inhibits target gene expression by an antigene mechanism. To simulate the kinetics of lac operon gene expression in the presence of antigene oligos, a genetically structured kinetic model, which includes transport of oligo into the cell, growth of bacteria cells, and lac operon gene expression, was developed. Predictions of the kinetic model fit the experimental data quite well after adjustment of the value of the oligonucleotide transport rate constant (9.0 x 10(-)(3) min(-)(1)) and oligo binding affinity constant (1.05 x 10(6) M(-)(1)). Our values for these two adjusted parameters are in the range of reported literature values.

Biological Transport↗

Assignment of ADA, ITPA, AK1, and AK2 to Chinese hamster chromosomes. Genetic and structural evidence for the conservation of mammalian autosomal synteny.

Interspecific somatic cell hybrids formed by fusion of mouse Cl1D cells with Chinese hamster primary spleen or fibroblast cells were used to form a clone panel with members retaining different combinations of Chinese hamster chromosomes and isozyme markers. Concordant segregation analysis of isozymes and chromosomes among clone panel members enabled the provisional assignment of ADA, ITPA, and AK1 to Chinese hamster chromosome 6. Diphtheria toxin selection for Chinese hamster chromosome 2 segregants in Dede cell X Cl1D somatic cell hybrids indicated AK2 is located on chromosome 2. These assignments extend observations of autosomal linkage group conservation in mammals--a concept further supported by similarity of G-band patterns in certain chromosomal regions bearing homologous loci in Chinese hamsters, humans, and mice.

Animals↗

Genetic and structural heterogeneity of proteophosphoglycans in Leishmania.

Proteophosphoglycans (PPG) are a large family of extensively glycosylated proteins with some unusual and unique features. The ppg gene family is conserved in at least three Leishmania species and localises to chromosome 35. Previous studies using standard discontinuous SDS-PAGE have been incapable of resolving PPG heterogeneity with most material failing to enter the resolving gel. We have exploited a continuous electrophoretic system, which allows for the first time the separation and characterisation of a low molecular weight population of PPG polypeptides. We provide evidence of surface expressed and developmentally regulated forms. Among those, we identify for the first time the previously described membrane-bound PPG and a form of filamentous fPPG, which is altered, or absent in two of the three L. major isolates examined.

Animals↗

Genetic population structure indicates sympatric speciation of Lake Malawi pelagic cichlids.

Allopatric processes of speciation have routinely been presented to explain the extraordinary radiation of the East African Great Lakes cichlid fish species flocks. The 21 or more species of pelagic cichlids within the Lake Malawi flock appear to have lake-wide distributions that challenge such a concept. Data from six microsatellite DNA loci indicate single, panmictic populations across the lake of three Diplotaxodon species. Levels of variability at these loci suggest that populations have been large and stable. Mitochondrial DNA sequence data (872 bp of control region + 981 bp of the NADH-2) from 90 species, representing all major clades within the Lake Malawi flock, indicate reciprocal monophyly of the pelagic clade. We suggest that these data support a hypothesis that speciation in sympatry is more plausible (and widespread) within the cichlid species flocks than previously thought.

Animals↗

Molecular genetics and structural biology of human MutT homolog, MTH1.

The human MTH1 gene located on chromosome 7p22 consists of 5 major exons. MTH1 gene produces seven types of mRNAs and the B-type mRNAs with exon 2b-2c segments direct synthesis of three forms of MTH1 polypeptides (p22, p21, and p18) by alternative initiation of translation, while the others encode only p18. In human cells, p18, the major form is mostly localized in the cytoplasm with some in the mitochondria. A single nucleotide polymorphism (SNP) in exon 2, which is tightly liked to another SNP (GTG83/ATG83), creates an additional alternative in-frame AUG in B-type MTH1 mRNAs yielding the fourth MTH1 polypeptide, p26 that possesses an additional mitochondrial targeting signal. These SNPs are likely to be one of the risk factors for cancer or for neuronal degeneration. The 30 amino acid residues are identical between MTH1 and MutT, and there is a highly conserved region consisting of 23 residues (MTH1: Gly36 to Gly58), with 14 identical residues. A chimeric protein in which the 23 residue sequence of MTH1 was replaced with that of MutT, retains the capability to hydrolyze 8-oxo-dGTP, indicating that the 23 residue sequences of MTH1 and MutT are functionally and structurally equivalent, and constitute a functional phosphohydrolase module. Saturated mutagenesis of the module in MTH1 indicated that an amphipathic property of the alpha-helix I consisting of 14 residues of the module (Thr44 to Gly58) is essential to maintain the stable catalytic surface for 8-oxo-dGTPase. MTH1 but not MutT efficiently hydrolyzes two forms of oxidized dATP, 2-hydroxy-dATP and 8-oxo-dATP, as well as 8-oxo-dGTP and 8-oxo-GTP. Thus, MTH1 is designated as the oxidized purine nucleoside triphosphatase and has a much wider substrate specificity than MutT. There is a significant homology between MTH1 protein and the C-terminal half of human MYH protein, which may be involved in the recognition of 8-oxoguanine and 2-hydroxyadenine.

Amino Acid Sequence↗

Genetic population structure and gene flow in the Atlantic cod Gadus morhua: a comparison of allozyme and nuclear RFLP loci.

High levels of gene flow have been implicated in producing uniform patterns of allozyme variation among populations of many marine fish species. We have examined whether gene flow is responsible for the limited population structure in the Atlantic cod, Gadus morhua L., by comparing the previously published patterns of variation at 10 allozyme loci to 17 nuclear restriction fragment length polymorphism (RFLP) loci scored by 11 anonymous cDNA clones. Unlike the allozyme loci, highly significant differences were observed among all populations at the DNA markers in a pattern consistent with an isolation-by-distance model of population structure. The magnitude of allele frequency variation at the nuclear RFLP loci significantly exceeded that observed at the protein loci (chi 2 = 24.6, d.f. = 5, P < 0.001). Estimates of gene flow from the private alleles method were similar for the allozymes and nuclear RFLPs. From the infinite island model, however, estimates of gene flow from the DNA markers were fivefold lower than indicated by the proteins. The discrepancy between gene flow estimates, combined with the observation of a large excess of rare RFLP alleles, suggests that the Atlantic cod has undergone a recent expansion in population size and that populations are significantly displaced from equilibrium. Because gene flow is a process that affects all loci equally, the heterogeneity observed among populations at the DNA level eliminates gene flow as the explanation for the homogeneous allozyme patterns. Our results suggest that a recent origin of cod populations has acted to constrain the extent of population differentiation observed at weakly polymorphic loci and implicate a role for selection in affecting the distribution of protein variation among natural populations in this species.

Animals↗

Genetic and structural analysis of the Rhizobium meliloti fixA, fixB, fixC, and fixX genes.

The fixA, fixB, fixC, and fixX genes of Rhizobium meliloti 1021 constitute an operon and are required for nitrogen fixation in alfalfa nodules. DNA homologous to the R. meliloti fixABC genes is present in all other Rhizobium and Bradyrhizobium species examined, but fixABC-homologous sequences were found in only one free-living diazotroph, Azotobacter vinelandii. To determine whether the fixABCX genes share sequence homology with any of the 17 Klebsiella pneumoniae nif genes, we determined the entire nucleotide sequence of the fixA, fixB, fixC, and fixX genes and defined four open reading frames that code for polypeptides of molecular weights 31,146, 37,786, 47,288, and 10,937, respectively. Neither DNA nor amino acid sequence homology to the R. meliloti fixA, -B, -C, and -X genes was found in the K. pneumoniae nif operon. The fixX gene contains a cluster of cysteine residues characteristic of ferredoxins and is highly homologous to an Azotobacter ferredoxin which has been shown to donate electrons to nitrogenase. The fixABC operon contains a promoter region that is highly homologous to other nifA-activated promoters. We also found a duplication of the 5' end of the fixABCX operon; a 250-bp region located 520 bp upstream of the fixABCX promoter bears more than 65% homology to the 5' end of the transcribed region, including the first 32 codons of fixA.

Amino Acid Sequence↗

Molecular population genetics of structural variants of esterase 6 in Drosophila melanogaster.

Several lines of evidence indicate that natural selection operates between the major EST6-F and EST6-S allozymes of Drosophila melanogaster. In particular, consistent latitudinal clines and seasonal variation in their relative frequencies strongly suggest that they are not selectively equivalent in field populations. Several laboratory studies have found frequency-dependent fitness differences among the Est6-F and Est6-S genotypes. Moreover, the purified EST6-F and EST6-S allozymes differ in biochemical properties and the physiology of the enzyme, as a major component of the seminal fluid, suggests that these differences could affect reproductive aspects of fitness. However, molecular analyses reveal high levels of variation in the EST6 protein both within and between the EST6-F and EST6-S allozymes. Limited thermostability and more sensitive electrophoretic analyses reveal at least 17 variants of the two allozymes and sequence comparisons among 13 isolates of the Est6 gene reveal 16 nucleotide polymorphisms that would lead to amino acid differences. Two closely linked amino acid differences are strongly associated with the major difference between EST6-F and EST6-S; either or both of these are likely to cause the observed biochemical differences between EST6-F and EST6-S and may be the primary targets for the selection between these allozymes. The functional and adaptive significance of the other amino acid polymorphisms is unclear, although the data suggest that the EST6-8 haplotype within EST6-S has both arisen and proliferated relatively recently.

Alleles↗

Mapping of the polA locus of Escherichia coli K12: genetic fine structure of the cistron.

The close linkage of the glnA gene with polA was exploited to construct a fine structure map of polA by means of generalized transduction with phage P1. Nine different polA- alleles were mapped by recombinational crosses. The results indicate a gene order consistent with previous observations (KELLEY and GRINDLEY 1976a; MURRAY and KELLEY 1979). Three mutations, polA5, polA6 and polA12 map within the "carboxy-terminal" or "large-fragment" portion of the gene in unambiguous order. Four alleles, known to affect the "amino-terminal" portion of the gene, polA107, polA214, polA480ex and polA4113, appear to be closely linked with certain ambiguities in their exact order. All four of these mutations are known to alter the 5' leads to 3' exonuclease activity of DNA polymerase I and three of them result in the conditional lethal polA- phenotype. The polA1 nonsense mutation maps between these two groups in a position consistent with its known effect, production of an amber fragment that includes the 5' leads to 3' exonuclease. The final allele, resA1, is another nonsense mutation that maps at the extreme "amino-terminus" of the cistron.----A number of control experiments were conducted to determine the effects of polA- mutations on the P1-mediated recombinational event. These experiments indicated that abortive transduction occurs quite frequently, but the formation of abortive transductants and segregation of unselected transduced markers among daughter progeny is like that observed by other investigators. There was no evidence that any individual polA- allele behaved in an exceptional fashion during recombination.

Alleles↗