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     

Identification of the development stage—specific factors in mouse fetal liver binding to the human β—globin gene promoter

In order to elucidate the molecular mechanisms of globin gene expression during embryonic development,the nuclear extracts from mouse hematopoietic tissue at different stages of development have been prepared.By using DNase I footprinting and gel mobility shift assays,the binding of protein factors in these extracts to the human β-globin promoter was analyzed.The differences in the binding patterns of protein factors during development were observed.An erythroid-specific and stage-specific nuclear protein in the nuclear extrace from d 18 mouse fetal liver was identified,which can bind to the sequence(from-66bp to-90bp) of human β-globin promoter.We therefore speculate that the function of this cis-acting element may be similar to stage selector element(SSE) in chicken β^A-promoter.     

The primary structure of the human ϵ-globin gene

We describe the complete nucleotide sequence of the human ?-globin gene including 387 nucleotides of 5′ flanking sequence and 301 nucleotides of 3′ flanking sequence. The arrangement of coding, noncoding and intervening sequences in this gene is entirely consistent with its identification as the embryonic β-like globin gene.     

Genomic organization and refined mapping of the mouse β-dystrobrevin gene

β-Dystrobrevin, a dystrophin-related protein that is expressed in non-muscle tissues, is highly homologous to α-dystrobrevin, a member of the dystrophin-associated protein complex (DPC). β-Dystrobrevin associates with Dp71 and syntrophin and is believed to have a role in non-muscle DPCs. Here we report the characterization and mapping of the mouse β-dystrobrevin gene. The mouse β-dystrobrevin gene is organized into 21 exons spanning over 130 kb of DNA. We provide evidence that this gene is transcribed from at least two promoter regions but appears to utilize a common translation initiation site. We show that the similarity between β-dystrobrevin and α-dystrobrevin is reflected in the conservation of their exon-intron junctions. β-Dystrobrevin has been localized to proximal mouse Chromosome (Chr) 12 by backcross mapping. A database search revealed that two mouse genetic diseases involving tissues expressing β-dystrobrevin have been mapped to this region, namely, congenital polycystic kidneys (cpk) and fatty liver dystrophy (fld). However, refined mapping analysis has excluded β-dystrobrevin as a candidate gene for either disease. Received: 1 June 1998 / Accepted: 16 July 1998     

The chromosomal distribution of repetitive DNA sequences within the human β globin gene cluster

Summary The chromosomal distribution and degree of repetitiveness of the sequences within the human globin gene cluster has been studied by in situ hybridisation. A genomic recombinant, H\G1, which contains 15.9 thousand base pairs (kb) of DNA inserted into charon 4A, was used as a template for the [³H] complementary RNA used for hybridisation. The inserted DNA contains sequences 4.7 kb to the 5 side of the gene and continues through the and genes to a site 2.8 kb to the 3 side of the gene. Two highly repetitive sequences, which are distributed evenly over all the chromosomes, have been identified within this DNA. Another somewhat less repetitive sequence has been identified between the and genes.     

Proteins binding to the 5‘—flanking regualtory elements of the human β—globin gene

The binding of nuclear proteins prepared from mouse erythroid tissue in different developmental stages to the 5‘-flanking regulatory elements of human β-globin gene,two negative control regions(NCR1,-610to-490 bp;NCR2,-338,to-233bp),was identified.Two stage specific protein factors corresponding to embryonic and fetal stages were found to be capable of binding to NCR2.These data provided evidence that the cis acting elements of the 5‘-flanking region might be involved in the developmental control of β-globin gene and NCR2 might be responsible in art for the silence of β-glolbin gene in the embryonic and fetal stages.     

Chromosomal arrangement of the chicken β-type globin genes

We have isolated the chicken β-type globin genes from a library of chicken DNA-λ Charon 4A recombinant bacteriophage. There are four β-type genes within this segment of the genome; we believe this represents all of the β-type genes of the chicken. The recombinant λCβG1 contains the embryonic ?- and adult β-globin genes. The hatching β^H and embryonic p-globin genes are found in the recombinant λCβG2. Although λCβG1 and λCβG2 do not physically overlap, we present evidence that all four genes are closely linked and transcribed from the same DNA strand. These experiments demonstrate that the chromosomal regions represented by λCβG1 and λCβG2 lie approximately 1.6 kb apart in the chicken genome. A third recombinant λCβG3 extends the genomic locus studied in the vicinity of the β-type globin genes to approximately 39 kb. The physical order of the chicken β-type globin genes within this segment of the chromosome is 5′ … ?-β^H-β-? … 3′. This arrangement is unique among the vertebrate β-type globin gene clusters thus far examined, in that embryonic genes are located at the 5′ and 3′ ends of the cluster while the hatching and adult genes occupy central positions.     

The organization of the β-globin gene of the bivalve mollusc Anadara trapezia and its evolutionary relationship to other invertebrate and vertebrate globin genes

Three pairs of oligonucleotide primers based on partial DNA and amino acid sequences were used in a combination of PCR experiments to amplify the -globin gene of the bivalve mollusc Anadara trapezia. The sequence of 2,139 by presented contains the whole of the -globin gene with the exception of the 5 flanking sequence. This gene possesses the three-exon-and-two-intron gene structure typical of vertebrate globin genes but the lengths of the introns (762 by and 690 bp, respectively) are only approximately half the size of those present in a -variant gene previously characterized from this organism. The encoded amino acid sequence shows two changes when compared to the previously published amino acid sequence. Correspondence to: A.G. Mackinlay     

A new base substitution in the 5′ regulatory region of the humanAγ globin gene is linked with the βs gene

A new TA base substitution, identified inside the 5 regulatory region of the human^A globin gene (^A –499 T A), is reported. This nucleotide change was found to be linked incis with the mutation producing sickle cell anemia (CD6 GAGGTG: ^s gene).     

The nucleotide sequence of the β-glucosidase gene from Cellvibrio gilvus

A gene encoding β-glucosidase from Cellvibrio gilvus, a cellobiose-producing bacterium, was cloned into Escherichia coli and sequenced. The structural gene consisted of 2565 bp encoding 854 amino acid residues with a characteristic signal peptide. A typical promoter sequence and SD region were located upstream of the initiation ATG codon. A sequence (180 amino acids) having high homology with those of β-glucosidases from several microorganisms was found in the deduced amino acid sequence of C. gilvus β-glucosidase. This sequence contains the aspartic acid residue which was found to be an active site residue in Aspergillus wentii β-glucosidase A3. The β-glucosidase gene of C. gilvus contains a high amount (69.4%) of G+C. These bases are localized not in the 3rd position of the codon, as is usually observed in G+C-rich genes, but rather in the 1st position. This result in a peptide which contains an extremely high amount (48%) of four amino acids (Pro, Ala, Arg, Gly) coded by CCN, GCN, CGN, and GGN.     

Sequence analysis of the upstream regions of Xenopus laevis β-globin genes and arrangement of repetitive elements within the globin gene clusters

The globin gene clusters of Xenopus laevis are interspersed by various different repetitive DNA elements. A specific repeat, the JH12 element, has been mapped by Southern analysis and some of its locations have been subsequently confirmed by nucleotide sequencing. JH12 family members seem to represent mobile genetic elements and display a high degree of divergence. The nucleotide sequences upstream to the adult _I-globin gene and to the two coordinately expressed larval _I- and _II genes have been determined and compared to those of the adult -genes. Besides some repetitive DNA elements and a short sequence of rather weak homology we have found no characteristic sequence motifs to be common to the adult - and -genes. The two larval -genes share one short sequence element being absent from the adult genes. This might reflect completely different sequence requirements for protein interactions and for the regulation of adult and larval globin gene expression.     

On the conformation of proteins: The handedness of the β-strand-α-helix-β-strand unit

The β-strand-α-helix-β-strand unit consists of two parallel, but not necessarily adjacent, β-strands which lie in a β-pleated sheet and are connected by one or more α-helices. This unit, which occurs in 17 functionally different globular proteins, may adopt a right- or a left-handed conformation. An analysis of the distribution shows that 57 out of the 58 units are right-handed. If the unit had no right-handed preference, the probability of observing such a distribution by chance is 10^?16. This may be explained in terms of the twist of the β-sheet which is shown to favour a right-handed unit, as otherwise steric hindrance occurs in the loop regions. We show that the right-handed strand-helix-strand unit determines the sense of the super-secondary structure found in the dehydrogenases and of related folds found in other structures. The evolutionary relationships between proteins containing this unit are re-evaluated in terms of this preference. The high probability that the unit will fold with a right-handed conformation has implications for the prediction of tertiary structure.     

Determination of the primary sequence of the duck αD globin mRNA and comparison of all adult duck and chick globin mRNA sequences

The nucleotide sequence of the duck α^D globin mRNA was determined. Its main feature is an exceptionally short 3′ non-coding segment of only 46 nucleotides, placed after the coding sequence of 141 codons. The last of the 6 adult globin mRNA of duck and chicken being thus sequenced, a comparison of all their features has become possible. Comparing the duck α^D mRNA to the related sequence in the chicken, we found greater homology than comparing it to the linked α^A globin sequence in the same species. Extensive homology can be found for a same globin chain α^A, α^D or β in between different avian species including also the goose and the ostrich; the avian α globin chains show a lower degree of sequence conservation in between species than the β chains. In contrast, within one species the three globin sequences have further diverged. The divergence between the α^A and α^D globin within a same species point to individual functional specificity and hence independent evolution and suggest that a mechanism of ‘gene conversion’ did not operate in between the avian α globin genes. Two segments of the amino acid sequence which we named ‘A^α’ and ‘B^α’ remain homologous in all avian α globins; two other regions ‘A^β’ and ‘B^β’ are identical in between the β globins. Segment A is placed at the 5′ end of exon II, and segment B at the 3′ end of the same exon; some amino acids in those segments are involved in the Heme binding site. Being almost identical in all know mammalian and avian globins of the α respectively the β type, regions A and B seem to represent the best conserved sequences in adult globin mRNA maintained during the divergence of species.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Targeted disruption of the mouse phospholipase C β3 gene results in early embryonic lethality

In order to investigate the biological function of phosphatidylinositol-specific phospholipase C (PLC) we generated mutant mice by gene targeting. Homozygous inactivation of PLCβ₃ is lethal at embryonic day 2.5. These mutants show poor embryonic organization as well as reduced numbers of cells. Identical phenotypes were recorded in homozygous mutants generated from two independently targeted embryonic stem cell clones. Heterozygous mutant mice, however, are viable and fertile for at least two generations. We also showed that mouse PLCβ₃ is expressed in unfertilized eggs, 3-cell and egg cylinder stages of embryos. In conclusion, these results indicate that PLCβ₃ expression is essential for early mouse embryonic development.