Contribution of gene conversion in the evolution of the human β-like globin gene family

Gene conversion is referred to as one of two types of mechanisms known to act on gene families, mainly to maintain their sequence homogeneity or, in certain cases, to produce sequence diversity. The concept of gene conversion was established 20 years ago by researchers working with fungi. A few years later, gene conversion was also observed in the human genome, i.e. the γ-globin locus. The aim of this article is to emphasize the role of genetic recombination, particularly of gene conversion, in the evolution of the human β-like globin genes and further to summarize its contribution to the convergent evolution of the fetal globin genes. Finally, this article attempts to re-examine the origin and spread of specific mutations of the β-globin cluster, such as the sickle cell or β-thalassemia mutations, on the basis of repeated gene conversion events. Received: 13 February 1997 / Accepted: 15 May 1998     

Characterization of β-casein gene in Indian riverine buffalo

The study aimed at characterization of buffalo β-casein gene and its promoter by PCR-SSCP analysis. Complete β-casein exon VII region analysis revealed two SSCP band patterns, with pattern-I representing predominant allele B (85%) present in homozygous (genotype BB) condition and pattern-II representing a rare allele A1 present in heterozygous condition (genotype A1B). Sequencing of two patterns revealed three nucleotide substitutions at codon 68, 151 and 193 of exon VII. The cDNA sequence of buffalo β-casein gene indicated three further nucleotide substitutions between allele A1 and B at codon 10, 39, and 41. Analysis of β-casein proximal promoter region (− 350 upstream to + 32) revealed four SSCP band patterns. These SSCP patterns corresponded to nucleotide substitutions at seven locations within 382 bp 5′ UTR region of β-casein gene. Haplotype analysis suggested pattern-I of exon VII (wild type) was associated with three types of promoters and pattern-II of exon VII (rare type) corresponded to one exclusive type of promoter. The study suggested two haplotypes of exon VII and four haplotypes of promoter for buffalo β-casein.     

Evaluation of seed storage-protein gene 5′ untranslated regions in enhancing gene expression in transgenic rice seed

5′ untranslated regions (UTRs) are important sequence elements that modulate the expression of genes. Using the β-glucuronidase (GUS) reporter gene driven by the GluC promoter for the rice-seed storage-protein glutelin, we evaluated the potential of the 5′-UTRs of six seed storage-protein genes in enhancing the expression levels of the foreign gene in stable transgenic rice lines. All of the 5′-UTRs significantly enhanced the expression level of the GluC promoter without altering its expression pattern. The 5′-UTRs of Glb-1 and GluA-1 increased the expression of GUS by about 3.36- and 3.11-fold, respectively. The two 5′-UTRs downstream of the Glb-1, OsAct2 and CMV35S promoters also increased GUS expression level in stable transgenic rice lines or in transient expression protoplasts. Therefore, the enhancements were independent of the promoter sequence. Real-time quantitative RT-PCR analysis showed that the increase in protein production was not accompanied by alteration in mRNA levels, which suggests that the enhancements were due to increasing the translational efficiencies of the mRNA. The 5′-UTRs of Glb-1 and GluA-1, when combined with strong promoters, might be ideal candidates for high production of recombinant proteins in rice seeds.     

Identification by gene deletion analysis of barS2, a gene involved in the biosynthesis of γ-butyrolactone autoregulator in Streptomyces virginiae

Virginiae butanolide (VB) is a member of the γ-butyrolactone autoregulators and triggers the production of streptogramin antibiotics virginiamycin M₁ and S in Streptomyces virginiae. A VB biosynthetic gene (barS2) was localized in a 10-kb regulatory island which controls the virginiamycin biosynthesis/resistance of S. virginiae, and analyzed by gene disruption/complementation. The barS2 gene is flanked by barS1, another VB biosynthetic gene catalyzing stereospecific reduction of an A-factor-type precursor into a VB-type compound, and barX encoding a pleiotropic regulator for virginiamycin biosynthesis. The deduced product of barS2 possessed moderate similarity to a putative dehydrogenase of Streptomyces venezuelae, encoded by jadW ₂ located in similar gene arrangement to that in the regulatory island of S. virginiae. A barS2-disruptant (strain IC152), created by means of homologous recombination, showed no differences in growth in liquid medium or morphology on solid medium compared to a wild-type strain, suggesting that BarS2 does not play any role in primary metabolism or morphological differentiation of S. virginiae. In contrast, no initiation of virginiamycin production or VB production was detected with the strain IC152 until 18 h of cultivation, at which time full production of virginiamycin occurs in the wild-type strain. The delayed virginiamycin production of the strain IC152 was fully restored to the level of the wild-type strain either by the exogenous addition of VB or by complementation of the intact barS2 gene, indicating that the lack of VB production at the initiation phase of virginiamycin production is the sole reason for the defect of virginiamycin production, and the barS2 gene is of primary importance for VB biosynthesis in S. virginiae. An erratum to this article can be found at     

Allelic characteristics of the β-amylase gene of barley varieties in Ukraine

Allelic diversity in a set of 99 spring and winter barley varieties intended for different use (malting, cereal, and valuable) was studied. PCR analysis with the use of the β-amylase DNA marker showed that genotypes of different barley varieties may include different alleles of the β-amy1 gene.     

A high proportion of mutations in the BRCA1 gene in German breast/ovarian cancer families with clustering of mutations in the 3′ third of the gene

We have analyzed 61 German breast and breast/ovarian cancer families for BRCA1 mutations using single-strand conformation polymorphism analysis (SSCP) followed by sequencing. Forty-seven of the families had at least three cases (at least two under 60 years) and 14 families had only two cases of breast/ovarian cancer (at least one under 50 years). Twenty-eight families were breast/ovarian and 33 were breast cancer-only families. Eighteen mutations in BRCA1 were detected in 11/28 breast/ovarian cancer families and 7/33 breast cancer families and none in the families with only two cases. We identified 17 truncation mutations (8 frameshift, 7 nonsense and 2 splice variants) and one missense mutation. Seven of these are novel and two, the 5382insC and 5622C→T mutations, occurred in two apparently unrelated families. The genotype of the two families with the 5382insC mutation is compatible with the rare haplotype segregating with the 5382insC mutation in different populations, further supporting its European origin. One unclassified missense alteration, R841W, was found in one family but did not segregate with the disease, suggesting that it is more likely a polymorphism. We also report and discuss the sequence of several new unclassified single-nucleotide changes first identified by SSCP. Of the 18 mutations, 13 occurred in the 3′ third of the gene (end of exon 11–24) and ovarian cancers were found in eight of these families. Received: 5 February 1998 / Accepted: 7 April 1998     

Genome-wide investigation of gene–environment interactions in colorectal cancer

Colorectal cancer (CRC), one of the most frequent neoplasias worldwide, has both genetic and environmental causes. As yet, however, gene–environment (G × E) interactions in CRC have been studied mostly for a small number of candidate genes only. Therefore, we investigated the possible interaction, in CRC etiology, between single-nucleotide polymorphisms (SNPs) on the one hand, and overweight, smoking and alcohol consumption on the other, at a genome-wide level. To this end, we adopted a two-tiered approach comprising a case-only screening stage I (314 cases) and a case–control validation stage II (259 cases, 1,002 controls). Interactions with the smallest p value in stage I were verified in stage II using multiple logistic regression analysis adjusted for sex and age. In addition, we specifically studied known CRC-associated SNPs for possible G × E interactions. Upon adjustment for sex and age, and after allowing for multiple testing, however, only a single SNP (rs1944511) was found to be involved in a statistically significant interaction, namely with overweight (multiplicity-corrected p = 0.042 in stage II). Several other G × E interactions were nominally significant but failed correction for multiple testing, including a previously reported interaction between rs9929218 and alcohol consumption that also emerged in our candidate SNP study (nominal p = 0.008). Notably, none of the interactions identified in our genome-wide analysis was with a previously reported CRC-associated SNP. Our study therefore highlights the potential of an “agnostic” genome-wide approach to G × E analysis.     

Expression of mouse α-amylase gene in methylotrophic yeastPichia pastoris

The expression of the mouse α-amylase gene in the methylotrophic yeast,P. pastoris was investigated. The mouse α-amylase gene was inserted into the multi-cloning site of a Pichia expression vector, pPIC9, yielding a new expression vector pME624. The plasmid pME624 was digested withSalI orBglII, and was introduced intoP. pastoris strain GS115 by the PEG1000 method. Fifty-three transformants were obtained by the transplacement of pME624 digested withSalI orBglII into theHIS ₄ locus (38 of Mut⁺ clone) or into theAOX1 locus (45 of Mut^s clone). Southern blot was carried out in 11 transformants, which showed that the mouse α-amylase gene was integrated into thePichia chromosome. When the second screening was performed in shaker culture, transformant G2 showed the highest α-amylase activity, 290 units/ml after 3-day culture, among 53 transformants. When this expression level of the mouse α-amylase gene is compared with that in recombinantSaccharomyces cerevisiae harboring a plasmid encoding the same mouse α-amylase gene, the specific enzyme activity is eight fold higher than that of the recombinantS. cerevisiae.     

Cloning of the β2-microglobulin gene in the zebrafish

The ₂-microglobulin (₂m) is a protein found in the serum in a free form and on the cell surface in a form noncovalently associated with the chain of the class I major histocompatibility complex (Mhc) molecules. In mammals, the ₂m-encoding gene (B2m) is found on a chromosome different from the Mhc proper. We have isolated and characterized the B2m gene of the zebrafish, Brachydanio rerio, family Cyprinidae. We obtained both cDNA and genomic clones of the Brre-B2m gene. The cDNA clones contained the entire coding sequence, the entire 3 untranslated (UT) region, and at least part of the 5UT region. The genomic clone contained the entire Brre-B2m gene. The coding sequence specifies 97 amino acid residues of the mature protein so that the zebrafish ₂m is two residues shorter than human and one residue shorter than cattle, fowl, or turkey ₂m (codons at positions 85 and 86 have been deleted in the Brre-B2m. gene). The amino acid and nucleotide sequence similarities between zebrafish and human ₂m (B2m) are 45% and 59%, respectively. Approximately 24% of the positions are invariant and an additional 9% show only conservative substitutions in comparisons which include all known 2m sequences (fish, avian, and mammalian). Most of the conserved positions are in the strands (some 47% of the -strand positions are conserved in the three vertebrate classes). The Brre-B2m gene consists of four exons separated by three introns. All of the introns are considerably shorter than the corresponding introns in the mammalian B2m genes. The coding sequences of the cDNA and the genomic clones are almost identical but the sequences of the 3'UT regions differ at 1.7% of the sites, suggesting that the genes borne by these clones might have diverged at least 0.7 million years (my) ago. In contrast to the human B2m gene, the Brre-B2m gene shows no bias in the distribution of the CpG dinucleotides: the dinucleotides are distributed evenly along the entire available sequence. The haploid genome of the zebrafish contains only one copy of the B2m gene.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L05383 (B2M) and L05384 (B2RG). Correspondence to: J. Klein.     

Comparison of theα-globin gene cluster structure in Perissodactyla

Summary To investigate molecular evolution in a mammalian order with a comprehensive fossil record, we have constructed-globin-like gene cluster maps for members of the order Perissodactyla. Although the arrangement of genes is the same in the five Equidae examined, the tapir and rhinoceros differ from each other and the horse in the position and number of their genes, but not in the arrangement of their and genes. In contrast to morphological work, a dendrogram derived from restriction site maps associates the tapir with the horse rather than with the rhinoceros; however, this phylogeny is not statistically significant. Among the Equidae,Equus caballus emerges as an outgroup, in agreement with data from other disciplines.