Gene evolution in the chicken β-globin cluster

We have determined the cDNA sequence of the chicken embryonic β-like ?-globin gene. Comparison with the sequences of the chicken ρ-globin and β-globin genes reveals the presence of two regions that are identical or nearly identical in ? and ρ. The first contains the 5′ untranslated sequence and exon 1, while the second region includes the second half of axon 2. Outside these regions ρ and ? are less homologous to each other than to the adult β-globin gene. The embryonic ρ and ? genes are located at opposite ends of the β-globin-gene cluster, not contiguously as are all other known pairs of simultaneously expressed globin genes. We suggest a role for gene conversion in the synchronization of expression of two highly diverged genes.     

Comparative genomic analysis of the proteasome β5t subunit gene: implications for the origin and evolution of thymoproteasomes

The thymoproteasome is a recently discovered, specialized form of 20S proteasomes expressed exclusively in the thymic cortex. Although the precise molecular mechanism by which the thymoproteasome exerts its function remains to be elucidated, accumulating evidence indicates that it plays a crucial role in positive selection of T cells. In the present study, we analyzed the evolution of the β5t subunit, a β-type catalytic subunit uniquely present in thymoproteasomes. The gene coding for the β5t subunit, designated PSMB11, was identified in the cartilaginous fish, the most divergent group of jawed vertebrates compared to the other jawed vertebrates, but not in jawless vertebrates or invertebrates. Interestingly, teleost fish have two copies of apparently functional PSMB11 genes, designated PSMB11a and PSMB11b, that encode β5t subunits with distinct amino acids in the S1 pocket. BLAST searches of genome databases suggest that birds such as chickens, turkey, and zebra finch lost the PSMB11 gene, and have neither thymoproteasomes nor immunoproteasomes. In mammals, reptiles, amphibians, and teleost fishes, the PSMB11 gene (the PSMB11a gene in teleost fish) is located next to the PSMB5 gene coding for the β5 subunit of the standard 20S proteasome, indicating that the PSMB11 gene arose by tandem duplication from the evolutionarily more ancient PSMB5 gene. The general absence of introns in PSMB11 and an unusual exon–intron structure of jawed vertebrate PSMB5 suggest that PSMB5 lost introns and duplicated in tandem in a common ancestor of jawed vertebrates, with PSMB5 subsequently gaining two introns and PSMB11 remaining intronless.     

A genome-wide screen of gene–gene interactions for rheumatoid arthritis susceptibility

The objective of the study was to identify interacting genes contributing to rheumatoid arthritis (RA) susceptibility and identify SNPs that discriminate between RA patients who were anti-cyclic citrullinated protein positive and healthy controls. We analyzed two independent cohorts from the North American Rheumatoid Arthritis Consortium. A cohort of 908 RA cases and 1,260 controls was used to discover pairwise interactions among SNPs and to identify a set of single nucleotide polymorphisms (SNPs) that predict RA status, and a second cohort of 952 cases and 1,760 controls was used to validate the findings. After adjusting for HLA-shared epitope alleles, we identified and replicated seven SNP pairs within the HLA class II locus with significant interaction effects. We failed to replicate significant pairwise interactions among non-HLA SNPs. The machine learning approach “random forest” applied to a set of SNPs selected from single-SNP and pairwise interaction tests identified 93 SNPs that distinguish RA cases from controls with 70% accuracy. HLA SNPs provide the most classification information, and inclusion of non-HLA SNPs improved classification. While specific gene–gene interactions are difficult to validate using genome-wide SNP data, a stepwise approach combining association and classification methods identifies candidate interacting SNPs that distinguish RA cases from healthy controls.     

Evolution of the β-globin gene cluster in man and the primates

The arrangement of primate β-related globin genes has been determined by restriction endonuclease mapping of genomic DNA from species ranging from prosimians to man. The arrangement of the entire ?γγδβ-globin gene cluster in the gorilla and the yellow baboon is indistinguishable from that of man. Restriction site differences between these species are consistent with a surprisingly low overall rate of intergenic DNA sequence divergence of approximately 1% in 5 million years. A new world monkey (owl monkey) has a single γ-globin gene, suggesting that the ^Gγ-^Aγ-globin gene duplication in man is ancient, and occurred about 20 to 40 million years ago. The β-globin gene cluster in the brown lemur, a prosimian, is remarkably short (about 20,000 base-pairs) and contains single ?-, γ- and β-globin genes. The γ- and β-globin genes in this animal are separated by a curious gene containing the 3′ end of a β-globin gene preceded by sequences related to the 5′ end of the ?-globin gene.     

A genome-wide Drosophila screen for heat nociception identifies α2δ3 as an evolutionarily conserved pain gene

Worldwide, acute, and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knockdown in Drosophila, we report a global screen for an innate behavior and identify hundreds of genes implicated in heat nociception, including the α2δ family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain-evoked signals from the thalamus to higher-order pain centers. Intriguingly, in α2δ3 mutant mice, thermal pain and tactile stimulation triggered strong cross-activation, or synesthesia, of brain regions involved in vision, olfaction, and hearing.     

Reproductive tissue-specific expression profiling and genetic variation across a 19 gene bovine β-defensin cluster

β-defensins are small cationic peptides, with potent immunoregulatory and antimicrobial activity which are produced constitutively and inducibly by eukaryotic cells. This study profiles the expression of a cluster of 19 novel defensin genes which spans 320 kb on chromosome 13 in Bos taurus. It also assesses the genetic variation in these genes between two divergently selected cattle breeds. Using quantitative real-time PCR (qRT-PCR), all 19 genes in this cluster were shown to be expressed in the male genital tract and 9 in the female genital tract, in a region-specific manner. These genes were sequenced in Norwegian Red (NR) and Holstein-Friesian (HF) cattle for population genetic analysis. Of the 17 novel single nucleotide polymorphisms (SNPs) identified, 7 were non-synonymous, 6 synonymous and 4 outside the protein coding region. Significant frequency differences in SNPs in bovine β-defensins (BBD) 115, 117, 121, and 122 were detected between the two breeds, which was also reflected at the haplotype level (P < 0.05). There was clear segregation of the haplotypes into two blocks on chromosome 13 in both breeds, presumably due to historical recombination. This study documents genetic variation in this β-defensin gene cluster between Norwegian Red and Holstein-Friesian cattle which may result from divergent selection for production and fertility traits in these two breeds. Regional expression in the epididymis and fallopian tube suggests a potential reproductive-immunobiology role for these genes in cattle.     

Structure of the chicken interferon-γ gene,and comparison to mammalian homologues

The sequence of the chicken interferon-γ (ifn-γ) gene was determined, one of the first non-mammalian cytokine gene structures to be elucidated. Initial genomic clones were amplified from chicken genomic DNA and were used to isolate a cosmid clone covering the entire gene for sequencing. The exon:intron structure of chicken ifn-γ is very similar to those of its mammalian homologues, with the exception of the third intron, which is markedly shorter in the chicken. The first exon contains both 5′ UTR and signal sequence and the first 22 aa of the mature protein. The remainder of the coding region lies in exons 2–4. Exon 4 also encodes the stop codon and the 3′ UTR, including two possible polyadenylation signals. A number of potential regulatory sequences similar to those found in mammals have been identified, in the promoter, in each intron and in the 3′ UTR. In the promoter, these include the TATAATA- and CCAT-boxes, a consensus GATA motif in the reverse orientation and a potential NF-κB binding site. Other regulatory elements identified in the promoters of mammalian ifn-γ genes are absent. Internal to the gene structure, regulatory sequences identified include elements found in the DNase I hypersensitivity region of the first intron of the human ifn-γ gene and several potential NF-κB binding sites. The 3′ UTR contains an AT-rich sequence, including nine repeats of the `instability' motif ATTTA. As in mammals, chicken ifn-γ is a single copy gene. The gene is highly conserved, with no polymorphisms yet identified using either RFLP or SSCP in the coding region. However, promoter sequence polymorphisms between different inbred lines of chickens have been identified, with possible links to disease resistance.     

A “GC-rich” method for mammalian gene expression:A dominant role of non-coding DNA GC content in the regulation of mammalian gene expression

High mammalian gene expression was obtained for more than twenty different proteins in different cell types by just a few laboratory scale stable gene transfections for each protein.The stable expression vectors were constructed by inserting a naturally-occurring 1.006 kb or a synthetic 0.733 kb DNA fragment(including intron) of extremely GC-rich at the 5' or/and 3' flanking regions of these protein genes or their gene promoters.This experiment is the first experimental evidence showing that a non-coding ex...     

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     

β-Thalassemia major resulting from a compound heterozygosity for the β-globin gene mutation: further evidence for multiple origin and migration of the thalassemia gene

Summary We describe in a Japanese family -thalassemia resulting from a compound heterozygosity for a -globin gene mutation. One mutation is a C-to-T transition at IVS-2 nucleotide position 654 on the background of Mediterranean haplotype IX. Another mutation is a G-to-A transition at IVS-2 nucleotide position 1, associated with a novel haplotype XL The occurrence of these mutations on various chromosomal backgrounds provides strong evidence for an interplay of gene migration, interallelic gene conversion, and multiple origins of the same mutation.     

Analysis of β-globin gene haplotypes in Asian Indians: origin and spread of β-thalassaemia on the Indian subcontinent

-globin gene haplotypes were determined for 196 normal (-A) and 419 thalassaemia (-Th) chromosomes of individuals from four different regions of the Indian subcontinent; North-west Pakistan, Gujarat, Punjab and Sindh. Analysis of -A and -Th haplotypes and haplotype-mutation associations in each regional group along with a consideration of Indian history provided information about the origin and spread of -thalassaemia mutations on the Indian subcontinent. The data are consistent with relatively recent and local origins for most -thalassaemia mutations. The frequencies of particular alleles differ markedly in various regions and these may be useful population markers. Of the high frequency alleles, intervening sequence 1 (IVS-1) nucleotide 5 (G-C) and codons 41/42 (-CTTT) appear to be older as suggested by multiple haplotype associations and a widespread geographical distribution. The microepidemiology of -thalassaemia in this region reflects considerable ethnic diversity, gene flow from population migration and natural selection by malaria infection.     

Polygynandry in a red fox population: implications for the evolution of group living in canids?

Canid social groups are typically thought to consist of extendedfamilies, that is, a dominant breeding pair and related nonbreedingsubordinates, that principally obtain indirect fitness benefitsfrom helping to raise the offspring of the dominant pair. Consequently,the monogamous pair has been viewed as the basic fundamentalunit of canid social organization. However, there have beenfew genetic studies that have tested this assumption. We analyzedthe parentage of red foxes (Vulpes vulpes) in a high-density(19.6–27.7 adult foxes/km²) population in Bristol, UK,to determine (1) whether groups typically produced a singlelitter of cubs annually and (2) whether male and female foxesexhibited monogamous mating strategies. Social monogamy (theproduction of one litter in a social group) was observed orassumed in 54% of breeding attempts (N = 13 group-years). However,polyandrous and polygynous patterns of mating were common. Multiplepaternity was confirmed in 38% of litters (N = 16) containingoffspring with resolved maternity and paternity (N = 30 cubs);when including cubs with unresolved paternity (N = 20), multiple-paternitymay have occurred in 69% of litters. Litters were sired by anaverage of 1.6 identified males (range = 1–4); when includingcubs with unresolved paternity, litters may have been siredby up to seven males. Only 20% (6/30) of cubs with resolvedmaternity and paternity were sired by males within the socialgroup. Within groups, dominant females did not breed with subordinatemales; dominant males did breed with subordinate females. Dominantand subordinate females both produced cubs with dominant andsubordinate males from other social groups. Mean adult relatednessin groups typically ranged from 0.15–0.35, indicativeof second-order rather than first-order relatives.     

A “GC-rich” method for mammalian gene expression: A dominant role of non-coding DNA GC content in regulation of mammalian gene expression

High mammalian gene expression was obtained for more than twenty different proteins in different cell types by just a few laboratory scale stable gene transfections for each protein. The stable expression vectors were constructed by inserting a naturally-occurring 1.006 kb or a synthetic 0.733 kb DNA fragment (including intron) of extremely GC-rich at the 5′ or/and 3′ flanking regions of these protein genes or their gene promoters. This experiment is the first experimental evidence showing that a non-coding extremely GC-rich DNA fragment is a super “chromatin opening element” and plays an important role in mammalian gene expression. This experiment has further indicated that chromatin-based regulation of mammalian gene expression is at least partially embedded in DNA primary structure, namely DNA GC-content.     

A high-throughput screen for the engineered production of β-lactam antibiotics

High-throughput screens and selections have had profound impact on our ability to engineer proteins possessing new, desired properties. These methods are especially useful when applied to the modification of existing enzymes to create natural and unnatural products. In an advance upon existing methods we developed a high-throughput, genetically regulated screen for the in vivo production of β-lactam antibiotics using a green fluorescent protein (gfp) reporter. This assay proved reliable and sensitive and presents a dynamic range under which a wide array of β-lactam architectural subclasses can be detected. Moreover, the graded response elicited in this assay can be used to rank mutant activity. The utility of this development was demonstrated in vivo and then applied to the first experimental investigation of a putative catalytic residue in carbapenem synthase (CarC). Information gained about the mutability of this residue defines one parameter for enzymatic activity and sets boundaries for future mechanistic and engineering efforts.     

The evolution of the α- and β-globin gene clusters in human populations

Summary DNA analysis of the - and -globin gene clusters has revealed substantial variability between individuals and populations. As well as restriction enzyme site and length polymorphisms, variation in gene copy number and type is observed. Because of this extensive polymorphism DNA analysis offers a highly informative method of studying genetic affinities between human populations. Haplotypes, consisting of a set of restriction enzyme polymorphisms distributed along the cluster, have been developed for both loci. Analysis of the molecular basis of numerous -thalassaemia alleles has revealed, in general, different sets of mutations in different populations, indicating that these postdate the racial divergence. Recent microepidemiological studies on the distribution of -thalassaemia support the hypothesis that this condition, like the {ie16-1}, has been selected because it confers protection against malaria. Population-specific DNA polymorphisms at these and other loci promise to be of considerable value to genetic anthropology.     

Characterization of a polymorphism in the 3′ part of the chicken vitellogenin gene

Summary An allele giving rise to a polymorphism within the 3 part of the chicken vitellogenin gene was cloned, sequenced, and compared to the previously cloned allele. The polymorphism is formed by a perfect copy of 343 bp from intron 32 in tandem array with a perfect copy of 244 bp from intron 33; this 587-bp element is inserted in a head-to-tail arrangement in intron 33. We propose a mechanism in which an unequal crossing-over resulted in a vitellogenin gene with two exons 33, one of which was subsequently deleted. Thus, intron 33 was enlarged by the tandem repeats without affecting the protein-encoding sequence of the gene. At the boundaries of the repeated elements, two short direct repeats are found that resemble the recombination signals of immunoglobulin genes. They may have had a key role in the formation of the new allele.