A new hypervariable marker for the human alpha-globin gene cluster.

We have located a highly polymorphic region of DNA approximately 100 kb upstream of the human alpha-globin genes (the alpha-globin 5' hypervariable region; 5'HVR). The element responsible is a minisatellite sequence comprising a variable copy number tandem repeat array of a G/C-rich 57-bp sequence. This increases the number of minisatellite elements in the vicinity of the alpha-globin genes to five, all of which share a region of sequence identity, thus raising questions concerning the distribution and origins of such tandem repeat sequences. The 5'HVR is highly polymorphic and, together with other hypervariable regions at this locus, provides a valuable genetic marker on the short arm of chromosome 16.     

Primate evolution of an olfactory receptor cluster: diversification by gene conversion and recent emergence of pseudogenes.

The olfactory receptor (OR) subgenome harbors the largest known gene family in mammals, disposed in clusters on numerous chromosomes. We have carried out a comparative evolutionary analysis of the best characterized genomic OR gene cluster, on human chromosome 17p13. Fifteen orthologs from chimpanzee (localized to chromosome 19p15), as well as key OR counterparts from other primates, have been identified and sequenced. Comparison among orthologs and paralogs revealed a multiplicity of gene conversion events, which occurred exclusively within OR subfamilies. These appear to lead to segment shuffling in the odorant binding site, an evolutionary process reminiscent of somatic combinatorial diversification in the immune system. We also demonstrate that the functional mammalian OR repertoire has undergone a rapid decline in the past 10 million years: while for the common ancestor of all great apes an intact OR cluster is inferred, in present-day humans and great apes the cluster includes nearly 40% pseudogenes.     

CpG islands from the alpha-globin gene cluster increase gene expression in an integration-dependent manner.

In contrast to other globin genes, the human and rabbit alpha-globin genes are expressed in transfected erythroid and nonerythroid cells in the absence of an enhancer. This enhancer-independent expression of the alpha-globin gene requires extensive sequences not only from the 5' flanking sequence but also from the intragenic region. However, the features of these internal sequences that are responsible for their positive effect are unclear. We tested several possible determinants of this activity. One possibility is that a previously identified array of discrete binding sites for known and potential regulatory proteins within the alpha-globin gene comprise an intragenic enhancer specific for the alpha-globin promoter, but directed rearrangements of the sequences show that this is not the case. Alternatively, the promoter may extend into the gene, with the function of the discrete binding sites being dependent on maintenance of their proper positions and orientations relative to the 5' flanking sequence. However, the positive effects observed in gene fusions do not localize to a discrete region of the alpha-globin gene and the results of internal deletions and point mutations argue against a required role of the targeted discrete binding sites. A third possibility is that the CpG island, which includes both the 5' flanking and intragenic regions associated with the positive activity, may itself have a more general effect on expression in transfected cells. Indeed, we show that the size of the CpG island in constructs correlates with the level of gene expression. Furthermore, the alpha-globin promoter is more active in the context of a previously inactive CpG island than in an A+T-rich context, showing that the CpG island provides an environment more permissive for expression. These effects are seen only after integration, suggesting a possible mechanism at the level of chromatin structure.     

Characterization of the major regulatory element upstream of the human alpha-globin gene cluster.

The major positive regulatory activity of the human alpha-globin gene complex has been localized to an element associated with a strong erythroid-specific DNase I hypersensitive site (HS -40) located 40 kb upstream of the zeta 2-globin mRNA cap site. Footprint and gel shift analyses of the element have demonstrated the presence of four binding sites for the nuclear factor GATA-1 and two sites corresponding to the AP-1 consensus binding sequence. This region resembles one of the major elements of the beta-globin locus control region in its constitution and characteristics; this together with evidence from expression studies suggests that HS -40 is a primary element controlling alpha-globin gene expression.     

Organisation and evolution of the tol-pal gene cluster

The genomic context and phylogenetic distribution of the tol-pal gene cluster and homologues to its various components have been investigated. The structure of this operon is well conserved across the gram negative bacteria, and the machine encoded by these genes probably evolved with the appearance of gram negative bacteria. Since the evolutionary appearance of the operon some species appear to have lost the genes. These bacteria seem to fall into two classes, namely obligate intracellular parasites and bacteria that produce large numbers of outer membrane vesicles. The evolution of the alphabeta and gamma proteobacteria was accompanied by the association of an additional gene (ybgC) with the operon. Several coincidences of genomic context argue for an important role of the tol-pal operon in cell envelope maintenance. Genes homologous to tolQ and tolR proved to be very widespread being found throughout the eubacteria, and one example in the archea, this distribution argues for an ancient origin of these genes. The genomic context of these genes often suggests a role in micronutrient uptake. Interestingly in all the cases examined the tolQ and tolR genes or their homologues appear to be present as a pair, with a potential for a tight translational regulation.     

Anthropogenetical analysis of abnormal human alpha-globin gene cluster arrangement on chromosome 16

An earlier study of human globin gene polymorphism in two Adriatic islands of Olib and Silba showed an abnormal arrangement of alpha-globin genes in two different individuals. The next step was to determine the degree of the kinship relationship between the two probands, one with a deleted and another with triplicated alpha-globin gene on the island Silba, and to determine the stability of this disorder through generations. We reviewed the parish registers (Status Animarum) of the island of Silba, dating from the year 1527, and constructed family trees for the two probands. Restriction endonuclease mapping was performed to study the arrangement of the alpha-globin genes in the offspring of our probands. A total of 183 ancestors completed the two family trees. The kinship relationship between them was established in the 5th, 6th, and 7th generation. The analysis of alpha-globin genes in the offspring of our probands showed the triplicated alpha-globin genes in two persons. We also found alpha-globin gene triplication in other three relatives. We did not find any deleted alpha-globin genes. We determined the kinship relationship between the two probands, one with deleted and the other with triplicated alpha-globin genes. This finding enabled us to determine the stability of this gene disarrangement through generations. It also showed new possibilities in anthropogenetic research, by combining the analyses of parish registers with those of modern genetic methods, such as restriction endonuclease mapping.     

Molecular characterisation of a hypervariable region downstream of the human alpha-globin gene cluster.

We have characterised an unusual, highly polymorphic region of DNA located 8-kb downstream of the human alpha-globin gene complex. This hypervariable region (alpha-globin 3' HVR) is composed of an array of 17-bp tandem repeats, the number of which differs considerably (70-450) from one allele to another. The sequence of the 17-bp repeats is highly conserved within and between alleles. Furthermore, this sequence identifies a core oligonucleotide [5'-GNGGGG(N)ACAG-3'] that is common to three previously characterised hypervariable regions. At reduced stringency, a probe to the 3' HVR detects a new family of multiallelic loci that will be of value in the study of human genetics.     

The distribution of CR1, and Alu-like family of interspersed repeats, in the chicken genome

We have studied the distribution of CR1, a family of short interspersed repeats, in the chicken genome; this family is homologous to the AluI family of man and to the B1-B2 families of mouse. Hybridization with a suitable probe showed that the vase majority of CR1 are located on the heaviest major component (1.708) of the genome which only represents 9% of chicken DNA. Some repeats were also found on the 1.702 and 1.704 components, but none on the 1.699 component (components are denoted by their buoyant densities in CsCl). The GC content of the repeats, 48%, matches that, 47%, of the major component mainly harboring it.     

Association of two DNA polymorphisms in the alpha-globin gene cluster: implications for genetic analysis.

A new polymorphic BgI II restriction endonuclease site in the alpha-globin gene complex has been found in Cypriot, Sardinian, and Greek populations. In all cases, this polymorphism is linked to a particular hypervariable region between the zeta 2 and zeta 1 genes. This suggests that these hypervariable regions are stable and will be useful for genetic analysis.     

Primate origins and the evolution of angiosperms

Traditionally, the morphological traits of primates were assumed to be adaptations to an arboreal way of life. However, Cartmill [1972] pointed out that a number of morphological traits characteristic of primates are not found in many other arboreal mammals. He contends that orbital convergence and grasping extremities indicate that the initial divergence of primates involved visual predation on insects in the lower canopy and undergrowth of the tropical forest. However, recent research on nocturnal primates does not support the visually-oriented predation theory. Although insects were most likely important components of the diets of the earliest euprimates, it is argued here that visual predation was not the major impetus for the evolution of the adaptive traits of primates. Recent paleobotanical research has yielded evidence that a major evolutionary event occurred during the Eocene, involving the angiosperms and their dispersal agents. As a result of long-term diffuse coevolutionary interactions with flowering plants, modern primates, bats, and plant-feeding birds all first arose around the Paleocene-Eocene boundary and became the major seed dispersers of modern tropical flora during the Eocene. Thus, it is suggested here that the multitude of resources available on the terminal branches of the newly evolved angiosperm, rain forest trees led to the morphological adaptations of primates of modern aspect.     

Chromosomal arrangement of the duck alpha-globin genes and primary structure of the embryonic alpha-globin gene pi

A recombinant lambda Charon 4A bacteriophage, D alpha G-1, carrying the genes coding for the duck embryonic (pi') and adult (alpha A, alpha D) alpha-like globins was isolated from a previously constructed duck DNA recombinant library. The three globin genes are transcribed from the same DNA strand and are arranged in the order of their expression during development: 5'-pi'-alpha D-alpha A-3'. We have determined the complete nucleotide sequence of the duck pi'-globin gene, including the flanking regions. Due to the unusual length of intron 1 (963 bp) and intron 2 (568 bp) the 2167-bp duck pi'-globin gene is by far the largest among all known mammalian or avian alpha- and beta-globin genes. For instance, the duck pi'-globin gene introns are almost twice as long as those of the chicken pi'-globin genes. A surprisingly high degree of nucleotide sequence homology (88%) has been found for the 5' flanking region (positions -1 to -223) of the duck and chicken pi'-globin gene.     

The distribution of CR1, an Alu-like family of interspersed repeats,in the chicken genome

We have studied the distribution of CR1, a family of short interspersed repeats, in the chicken genome; this family is homologous to the AluI family of man and to the B1-B2 families of mouse. Hybridization with a suitable probe showed that the vast majority of CR1 are located on the heaviest major component (1.708) of the genome which only represents 9% of chicken DNA. Some repeats were also found on the 1.702 and 1.704 components, but none on the 1.699 component (components are denoted by their buoyant densities in CsCl). The GC content of the repeats, 48%, matches that, 47%, of the major component mainly harboring it.     

A novel regulatory element of the human alpha-globin gene responsible for its constitutive expression

The alpha-globin gene is expressed at a constitutively high level upon gene transfer into both erythroid and nonerythroid cells. The beta-globin gene, on the other hand, is dependent on the presence of a linked viral enhancer for its efficient expression upon transfer into heterologous cells. In this report, we describe a novel regulatory element within the structural alpha-globin gene which can activate its own promoter to result in a high level of expression in both erythroid and non-erythroid cells. This regulatory element does not appear to have the properties of a classical enhancer. While this element exerts a positive effect on its own promoter, we have demonstrated in a previous study that the same element exerts a negative effect on heterologous genes such as the beta- and gamma-globin genes. In this study, we localize this element to a 259 nucleotide fragment immediately downstream from the translation initiation codon which is partially overlapped by a DNase I hypersensitive domain only in erythroid cells. We propose that this element may activate the alpha-globin gene promoter in all cell types in vivo as it does in vitro. The specificity of erythroid expression of the alpha-globin gene in vivo is probably determined by a "permissive" chromatin configuration in erythroid cells and a "nonpermissive" configuration in non-erythroid cells.