Adaptive evolution of α-amylase genes in wild barley (Hordeum spontaneum) on micro and macro scales

Alpha-amylases play essential roles in germination, and the malting and brewing processes, by hydrolyzing starch granules present in the endosperm of barley.Hordeum spontaneum C. Koch, the progenitor of cultivated barley that harbors rich genetic diversity, was collected from seven different environments. To investigate the influence of microclimatic ecological divergence on α-amylase, single nucleotide polymorphisms (SNPs) in amy genes from these populations were determined. A total of 16 and 17 SNPs were detected in the coding sequences of amy1 and amy2, respectively, from the seven wild barley populations. Among these SNPs, three in amy1-2 and nine in amy2-2 were significantly associated with ecological factors. The genetic divergence of amy sequences was significantly different among the populations. Natural microclimatic selection was apparently the major evolutionary driving force causing interslope divergence and adaptive evolution of these genes. The genetic variation in amy1-2 and amy2-2 was at least partly ecologically determined in these populations, representing adaptive patterns generated by natural selection. The SNPs were apparently generated by natural selection in climatic environmental patterns at both the micro (“Evolution Canyon”) and macro (across Israel, Galilee, and Negev) scales.     

The human amylase-encoding genes amy2 and amy3 are identical to AMY2A and AMY2B

Inspection of the published nucleotide sequences reveals that the human amylase-encoding genes, amy2 and amy3, must be identical to the genes AMY2A and AMY2B, respectively.     

Cloning and sequence comparison of the mouse, human, and chicken engrailed genes reveal potential functional domains and regulatory regions.

We have isolated and characterized genomic DNA clones for the human and chicken homologues of the mouse En-1 and En-2 genes and determined the genomic structure and predicted protein sequences of both En genes in all three species. Comparison of these vertebrate En sequences with the Xenopus En-2 [Hemmati-Brivanlou et al., 1991) and invertebrate engrailed-like genes showed that the two previously identified highly conserved regions within the En protein ]reviewed in Joyner and Hanks, 1991] can be divided into five distinct subregions, designated EH1 to EH5. Sequences 5' and 3' to the predicted coding regions of the vertebrate En genes were also analyzed in an attempt to identify cis-acting DNA sequences important for the regulation of En gene expression. Considerable sequence similarity was found between the mouse and human homologues both within the putative 5' and 3' untranslated as well as 5' flanking regions. Between the mouse and Xenopus En-2 genes, shorter stretches of sequence similarity were found within the 3' untranslated region. The 5' untranslated regions of the mouse, chicken and Xenopus En-2 genes, however, showed no similarly conserved stretches. In a preliminary analysis of the expression pattern of the human En genes, En-2 protein and RNA were detected in the embryonic and adult cerebellum respectively and not in other tissues tested. These patterns are analogous to those seen in other vertebrates. Taken together these results further strengthen the suggestion that En gene function and regulation has been conserved throughout vertebrate evolution and, along with the five highly conserved regions within the En protein, raise an interesting question about the presence of conserved genetic pathways.     

Regulatory regions in the yeast FBP1 and PCK1 genes.

By deletion analysis of the fusion genes FBP1-lacZ and PCK1-lacZ we have identified a number of strong regulatory regions in the genes FBP1 and PCK1 which encode fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase. Lack of expression of beta-galactosidase in fusions lacking sequences from the coding regions suggests the existence of downstream activating elements. Both promoters have several UAS and URS regions as well as sites implicated in catabolite repression. We have found in both genes consensus sequences for the binding of the same regulatory proteins, such as yAP1, MIG1 or the complex HAP2/HAP3/HAP4. Neither deletion nor overexpression of the MIG1 gene affected the regulated expression of the FBP1 or PCK1 genes.     

Evolution of genes for allelic and isotypic forms of immunoglobulin kappa chains and of the genes for T-cell receptor beta chains in rabbits

New insights into the evolution of the families of genes encoding immunoglobulins and T-cell receptors of rabbits (Oryctolagus cuniculus) have come from molecular genetic studies. In contrast to human and mouse, rabbits were shown to have two genes for the constant region of immunoglobulin light chains (C kappa 1 and C kappa 2 isotypes) and complex allelic variants of K1 (allotypes). Although K1 allotype protein sequences differed at up to 41% of the amino acid positions, 3' untranslated, 5', and 3' flanking regions were conserved, and in the coding regions 78-80% of the codons with differences had replacement changes. Proportions of silent changes and changes in noncoding regions were comparable. Thus, in spite of their markedly different protein sequences, the K1b4, b5, and b9 allotypes appeared to be products of allelic genes. Molecular genetic analyses suggested that they may have undergone rapid divergence after an ancestral K2-like gene duplicated. Some rabbits were found to have two similar T-cell receptor C beta genes as do humans and many strains of mice, but others appeared to have three different C beta. In addition, we found allotypic forms of C beta. Some of the C beta allotypic differences occurred at positions where analogous C kappa allotypic differences were found. We also found V beta in mouse and human that were more similar to rabbit V beta than closely linked rabbit genes were to each other. This contrasts with rabbit immunoglobulin VH gene sequences that reflect concerted evolution. The data suggested that T-cell receptor V beta genes duplicated prior to mammalian radiation.     

Latent a1 VH germline genes in an a2a2 rabbit. Evidence for gene conversion at both the germline and somatic levels

We have previously reported the sequences of putative latent a1 cDNA derived from an alpha 2 alpha 2 rabbit. Significant similarity to nominal a1 cDNA sequences was noted, but none of the latent sequences were completely a1-like. We have now probed a genomic library, produced from the same alpha 2 alpha 2 rabbit, for evidence of germline latent a1 VH genes. Four hundred ninety-four VH+ clones were screened with oligonucleotides specific for a1 diagnostic regions of framework region 1 (FR1) and FR3. Twenty-two percent of the VH+ clones hybridized with an a1FR3 oligonucleotide probe. Two a1 FR1 probes yielded weak signals with 6% to 13% of the VH+ clones. Twenty VH genes from clones positive for one or more of the a1-specific oligonucleotide probes were sequenced, revealing 14 unique germline VH genes. All but one of these genes were 85% to 92% identical to the VH1-a1 nominal gene prototype, with sequence identity extending into the leader intron. Most genes displayed extended regions of similarity to a1 in FR1, FR3, or both and expressed 13 to 17 of the 21 allotype-associated residues, consistent with the nominal a1 sequence. The a1-like sequences were variously interspersed with short non-a1 segments, suggestive of germline gene conversion. Although none of the germline a1-like VH genes we have isolated from the alpha 2 alpha 2 rabbits are identical to the known a1 genes or protein sequences from alpha 1 alpha 1 rabbits and 8 of 14 are pseudogenes, most could make significant contributions to the synthesis of a complete nominal a1 sequence by serving as a pool of sequence donors during somatic gene conversion.     

Primary structure of human pancreatic alpha-amylase gene: its comparison with human salivary alpha-amylase gene

We have determined the entire structure of the human pancreatic alpha-amylase (Amy2) gene. It is approx. 9 kb long and is separated into ten exons. This gene (amy2) has a structure very similar to that of human salivary alpha-amylase (Amy1) gene [Nishide et al. Gene 41 (1986a) 299-304] in the nucleotide sequence and the size and location of the exons. The major difference lies in the fact that amy1 has one extra exon on the 5' side. Other differences are at the 5' border of exon 1 and the 3' border of exon 10. The close similarity of these two genes, as compared with mouse pancreatic and salivary amylase genes, suggests that during evolution, the divergence into the two amylase genes may have occurred after the divergence of mice and man.     

Predominance of VH-D-JH junctions occurring at sites of short sequence homology results in limited junctional diversity in neonatal antibodies.

Sequence diversity at the junctions of Ig genes differs between newborn and adult mice in two respects: 1) fetal/newborn Ig lack N regions; and 2) these N- junctional sequences very often contain 1 to 6 nucleotides that could have been encoded by either of the two joined gene segments. We address the hypothesis that such short homologies preferentially direct recombination to that site, and we analyze the effect of such homology-directed recombination upon the neonatal Ig repertoire. We examined 546 CDR3 sequences that were generated from polymerase chain reaction-amplified DNA from fetal and newborn liver using primers from three different VH families: S107, 7183, and J558. All junctional sequences using 14 frequently occurring IgH V-D and D-J gene combinations were analyzed. In 12 of the 14 combinations analyzed, there were 1 to 3 short sequence homologies, and the junctional sequences that would be created by those homologies were observed with high frequency. The D-J junctions often had two to three predominant junctional sequences, whereas the V-D junctions had one dominant junctional sequence. The only exceptions were the VHJ558-D junctions, where homology-directed recombination using the sequence homology between VHJ558 genes and most D genes would result in an out-of-frame join, and most of our sequences were productive. This latter result further suggests that homology-directed recombination may play a role in the nonrandom VH gene usage observed in fetal and newborn mice. Thus, most neonatal IgH junctions show limited diversity, not only due to the lack of N regions, but also because of nonrandom junctional sequences. Inasmuch as the few adult N- junctions also show a high frequency of homology-directed junctional sequences, V-D-J recombination throughout life may involve pairing via short homologies, with addition of N regions obscuring its role in the formation of adult IgH junctions.     

Vitelline envelope genes of the yellow fever mosquito, Aedes aegypti

Vitelline envelope genes from the mosquito Aedes aegypti were analyzed with respect to their DNA sequences, genomic representation, temporal and spatial expression profiles and response to 20-hydroxyecdysone. Genomic clones of three vitelline envelope genes, 15a-1, 15a-2 and 15a-3 were isolated. Southern analysis indicates that all three genes are represented by a single copy in the genome. The deduced amino acid sequences of all three vitelline envelope genes contain a conserved region of 46 residues that overlaps with a region that is conserved in four Drosophila melanogaster vitelline envelope genes. DNA was sequenced flanking the 15a-1, 15a-2 and 15a-3 coding regions. A 360 bp sequence 5′ of the 15a-2 coding region was identified with 72% identity to a sequence upstream of the Ae. aegypti VgA1 vitellogenin gene. The temporal patterns of 15a-1, 15a-2 and 15a-3 expression, as determined by Northern analysis, were similar. The spatial patterns of expression, as determined by whole-mount in situ hybridization, differed between the three genes. 15a-1 and 15a-3 were only expressed in the middle and posterior regions of the follicle, while 15a-2 was also expressed at the anterior region. Vitelline envelope gene expression was higher in ovaries that were dissected at 0, 2 and 10 h following a blood meal and then incubated in vitro for 10 h in medium containing 10⁻⁵ M 20-hydroxyecdysone, compared to ovaries that were incubated without hormone.     

甘蔗乙烯合成酶基因家族三个成员的克隆与序列分析

ACC（1-aminocyclopropane-1-carboxylic acid）合成酶是高等植物乙烯生物合成途径中的限速酶.根据已克隆的植物ACS（1-aminocyclopropane-1-carboxylic acid synthase）基因同源序列,设计简并引物,以甘蔗叶片总DNA为模板,通过PCR扩增,得到3条特异性强的扩增片段：Sc-ACS1为1 041 bp、Sc-ACS2为1 345 bp和Sc-ACS3为1 707 bp.将序列在GenBank核酸数据库进行同源性搜索,结果表明,3个片段均为ACS基因,推导编码的蛋白质序列分别包含326、242和310个氨基酸.其中,Sc-A CS1和Sc-ACS3同源性最高,核苷酸序列和蛋白质氨基酸序列分别有98%和96%同源,与禾本科植物玉米Zm ACS6、水稻OS-ACS2、毛竹等ACS基因家族也有很高的同源性,核苷酸序列同源性为88%-98%,蛋白质氨基酸序列同源性为73%-81%.甘蔗Sc-ACS2与水稻OS-ACS5在核苷酸和氨基酸序列上分别有91%和79%同源性,但与甘蔗Sc-ACS1和Sc-ACS3基因成员之间,氨基酸同源性分别只有45%和49%.系统进化分析表明,Sc-ACS1和Sc-ACS3基因与玉米Zm ACS6基因亲缘关系最近,而Sc-ACS2基因与水稻OS-ACS5基因亲缘关系最近.Southern杂交表明三基因在基因组中确实存在而且是多拷贝基因.三个片段已在GenBank数据库中注册,注册号分别为AY620985、AY620986和AY788919.     

The relative positions of sea urchin histone genes on the chimeric plasmids pSp2 and pSp17 as studied by electron microscopy

The relative positions of the sea urchin histone genes and the spacer regions on the chimeric plasmids pSp2 and pSp17 have been mapped by hybridizing total histone messenger RNA to single strands of the plasmid DNAs. The lengths and spacing between the several RNA:DNA duplex regions on the single strands of DNA were measured by the gene 32-ethidium bromide electron microscope mapping method. We find that the genes are interdigitated with spacer sequences of different lengths; that there are three coding sequences on pSp2, all on the same strand, with the relative order H1, H4, and B4; and that there are two coding sequences on pSp17, both on the same strand, corresponding to the messages denoted B1 and B2–B3, where B4, B1, and B2–3 are electrophoretically resolved components of histone mRNA, all of size intermediate between the larger H1 and the smaller H4 message.