Complete nucleotide sequence of the rabbit beta-like globin gene cluster. Analysis of intergenic sequences and comparison with the human beta-like globin gene cluster

The nucleotide sequence of the entire beta-like globin gene cluster of rabbits has been determined. This sequence of a continuous stretch of 44.5 x 10(3) base-pairs (bp) starts about 6 x 10(3) bp upstream from epsilon (the 5'-most gene) and ends about 12 x 10(3) bp downstream from beta (the 3'-most gene). Analysis of the sequence reveals that: (1) the sequence is relatively A + T rich (about 60%); (2) regions with high G + C content are associated with OcC repeats, a short interspersed repeated DNA in rabbits; (3) the distribution of polypurines, polypyrimidines and alternating purine/pyrimidine tracts is not random within the cluster; (4) most open reading frames are associated with known globin coding regions, OcC repeats or long interspersed repeats (L1 repeats); (5) the most prominent open reading frames are found in the L1 repeats; (6) different strand asymmetries in base composition are associated with embyronic and adult genes as well as the tandem L1 repeats at the 3' end of the cluster; and (7) essentially all the repeats appear to have been inserted by a transposon mechanism. A comparison of the sequence with itself by a dot-plot analysis has revealed nine new members of the OcC family of repeats in addition to the six previously reported. The OcC repeats tend to be clustered, particularly in the epsilon-gamma and gamma-psi delta intergenic regions. Dot-plot comparisons between the rabbit and the human clusters have revealed extensive sequence matches. Homology starts about 6 x 10(3) bp 5' to epsilon or as far upstream as the rabbit sequence is available. It continues throughout the entire cluster and stops about 0.7 x 10(3) bp 3' to beta, at which point several repeats have inserted in both rabbits and humans. Throughout the gene cluster, the homology is interrupted mainly by insertions or deletions in either the rabbit or the human genome. Almost all of the insertions are of known short or long repeated DNAs. The positions of the insertions are different in the two gene clusters, which indicates that both short and long repeats have been transposing throughout the genome for the time since the mammalian radiation. An alignment of rabbit and human sequences allows the calculation of the substitution rate around epsilon. Sequences far removed from the gene are evolving at a rate equivalent to the pseudogene rate, although some short regions show an apparently higher rate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular cloning and characterization of the human beta-like globin gene cluster

The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.     

Structure and function of the enhancer 3'' to the human A gamma globin gene.

An enhancer is located immediately 3' to the A gamma globin gene. We have used DNase I footprinting to map the sites of interaction of nuclear proteins with the DNA sequences of this enhancer. Eight footprints were discovered, distributed over 600 base pairs of DNA. Three of these contain a consensus binding site for the erythroid specific factor GATA-I. Each of these GATA-1 sites had an enhancer activity when inserted into a reporter plasmid and tested in human erythroleukemia cells. Other footprints within the enhancer contained consensus binding sequences for the ubiquitous, positive regulatory proteins AP2 and CBP-1. An Sp1-like recognition sequence was also identified. Synthetic oligonucleotides encompassing two of the footprints generated a slowly migrating complex in gel mobility shift assays. The same complex forms on a fragment of the human gamma globin gene promoter extending from -260 to -200. The DNaseI footprint of this protein complex with the enhancer overlapped a sequence, AGGAGGA, found within the binding site for a protein that interacts with the chicken beta globin promoter and enhancer, termed the stage selector element. We propose that this complex of proteins may be involved in the human gamma globin promoter-enhancer interaction.     

Isolation of the chicken beta-globin gene and a linked embryonic beta-like globin gene from a chicken DNA recombinant library.

A library of random chicken DNA fragments, 15-22 kb long, has been prepared in the vector lambda Charon 4A. This library was screened with combined adult and embryonic globin cDNA, and several independent globin gene-containing recombinants were isolated. One of these recombinants, lambda Chicken beta-globin 1 (lambda C beta G1), contains the adult chicken beta-globin gene and a closely linked embryonic beta-like globin gene. Both genes are transcribed in the same direction with the adult gene located 5' to the embryonic gene. Electron microscopic visualization of R loop structures generated by hybridization of globin RNA to lambda C beta G1 demonstrates that both globin genes contain major intervening sequences about 800 bp long, similar to those present in mammalian beta-globin genes. The adult beta-globin gene also contains a minor (approximately 100 bp long) intervening sequence analogous to the one observed in mammalian beta-globin genes. Restriction enzyme analysis of the adult beta-globin gene on lambda C beta G1 is consistent with the hypothesis that its two intervening sequences occur in the same positions with respect to the beta-globin amino acid sequence as do the corresponding mammalian intervening sequences.     

Localization of the beta-like globin gene cluster and the genes for parathyroid hormone and c-Harvey-ras 1 to region q14----q21 of rabbit chromosome 1 by in situ hybridization

The rabbit beta-like globin gene cluster (HBBC), comprised of epsilon-, gamma-, delta-, and beta-globin genes (HBE, HBG, HBD, and HBB, respectively), has been mapped to chromosome 1, region q14----q21, by in situ hybridization using probes for rabbit HBE and HBG. Probes for the human parathyroid hormone gene (PTH) and the Harvey-ras 1 protooncogene (HRAS1) also localize to this region by in situ hybridization. Thus, these genes are syntenic in lagomorphs as well as four other mammalian orders (primates, rodents, carnivores, and artiodactyls). The mapping data in rabbits provide further evidence that this synteny is strongly conserved in the evolution of mammalian chromosomes.     

Block duplications of a zeta-zeta-alpha-theta gene set in the rabbit alpha-like globin gene cluster

In order to understand the coordinate regulation between the alpha-like and beta-like globins during the developmental switches in hemoglobin synthesis, we have studied the rabbit alpha-like globin gene family. A cluster of six linked genes arranged 5'-zeta 1-alpha 1-theta 1-zeta 2-zeta 3-theta 2-3' has been isolated as a set of overlapping clones from a library of rabbit genomic DNA. Blot-hybridization analysis of genomic DNA not only confirms this linkage arrangement but also reveals the presence of additional zeta and theta genes. We propose that this gene cluster was generated by a block duplication of a set of alpha-like genes; the proposed duplication unit is zeta-zeta-alpha-theta. Further duplications of a zeta-zeta-theta set are also proposed to have occurred. As expected for a duplicated locus, the rabbit alpha-like gene cluster contains long blocks of internal homology. The Z homology block is about 7.2 kilobase pairs long and contains the zeta genes; the T homology block is about 4.7 kilobase pairs long and contains a theta gene. Surprisingly, both Z and T homology blocks are flanked by a common junction sequence (J) which contains a region very similar to the 3'-untranslated sequence of an alpha-globin gene. Analysis of the J sequences suggests a recombination mechanism by which the alpha gene could have been deleted from the second set of genes in the cluster (zeta 2-zeta 3-theta 2). The relationships among the genes in characterized alpha-like gene clusters in mammals are summarized. The rabbit gene cluster differs from those of other mammals principally in the loss of a gene orthologous to the human psi alpha 1 and in the block duplication of the zeta-zeta-alpha-theta gene set.     

The Saccharomyces cerevisiae MET3 gene: nucleotide sequence and relationship of the 5'' non-coding region to that of MET25

Summary In Saccharomyces cerevisiae, the expression of several genes implicated in methionine biosynthesis is coregulated by a specific negative control. To elucidate the molecular basis of this regulation, we have cloned two of these genes, MET3 and MET25. The sequence of MET25 has already been determined (Kerjan et al. 1986). Here, we report the nucleotide sequence of the MET3 gene along with its 5 and 3 flanking regions. Plasmids bearing different deletions upstream of the transcribed region of MET3 were constructed. They were introduced into yeast cells and tested for their ability to complement met3 mutations and to respond to regulation by exogenous methionine. The regulatory region was located within a 100 bp region. The sequence of this regulatory region was compared with that of MET25. A short common sequence which occurs 250–280 bp upstream of the translation initiation codon of the gene was found. This sequence is a good candidate for the cis-acting regulatory element.     

Analysis of an inversion within the human beta globin gene cluster.

We have cloned and sequenced the DNA from two regions of the defective beta-globin gene cluster from a patient with Indian A gamma delta beta thalassaemia, and confirmed the complex and unusual pattern of rearrangement involving two separate deletions (0.8 kb and 7.5 kb) the inversion of the 15.5 kb segment separating them, as previously proposed from gene mapping studies [1]. All four breakpoints occur within the transcribed region of the globin genes and at one junction are found six nucleotides of unknown origin. This unique rearrangement results in enhanced expression of the upstream fetal gene, and is therefore is pertinent to the localisation of any putative control region involved in the coordinate expression of fetal and adult genes.     

A 6-bp deletion 5'' to the G gamma globin gene in beta S chromosomes bearing the Bantu haplotype.

Sequencing of the upstream region of a human G gamma gene linked to the Bantu haplotype revealed a 6-bp deletion between site -400 and -395. Further analysis revealed that this mutation is present in 37% of the sickle cell anemia patients bearing the Bantu haplotype and is absent in the other haplotypes linked to the beta S gene, as well as in most chromosomes bearing the beta A-globin gene. The most parsimonious interpretation of the data is that the deletion is a very recent event which occurred in the subset of Bantu chromosomes already bearing a gene conversion of the A gamma gene by the G gamma gene. Its presence in black beta S chromosomes is most probably the consequence of a crossing-over between a Bantu beta S chromosome (with deletion and gene conversion) and a beta A chromosome.     

Comparative analysis of the locus control region of the rabbit beta-like gene cluster: HS3 increases transient expression of an embryonic epsilon-globin gene.

The rabbit homolog to the locus control region (LCR) of the human beta-like globin gene cluster was isolated, and long segments containing the DNase I hypersensitive sites (HS) were sequenced. The order and spacing of HS4, HS3, HS2 and HS1 are conserved between rabbit and human. Alignment of these sequences with their homologs from human, goat, and mouse shows that very long segments of DNA match between species, for over a thousand base pairs on either side of the previously identified functional cores, indicating that some important functions are found outside the cores. The activity of rabbit HS2 and HS3 was tested by attaching each to a novel reporter gene constructed by inserting the luciferase coding region into the rabbit epsilon-globin gene. In contrast to previous reports showing no effect of human or mouse HS3 on transient expression, both the rabbit HS2 and HS3 DNA fragments separately increased transient expression from the epsilon-luciferase hybrid gene and expression from stably integrated constructs in K562 erythroleukemia cells.     

Characterization of CYP76M5-8 indicates metabolic plasticity within a plant biosynthetic gene cluster

Recent reports have revealed genomic clustering of enzymatic genes for particular biosynthetic pathways in plant specialized/secondary metabolism. Rice (Oryza sativa) carries two such clusters for production of antimicrobial diterpenoid phytoalexins, with the cluster on chromosome 2 containing four closely related/homologous members of the cytochrome P450 CYP76M subfamily (CYP76M5-8). Notably, the underlying evolutionary expansion of these CYP appears to have occurred after assembly of the ancestral biosynthetic gene cluster, suggesting separate roles. It has been demonstrated that CYP76M7 catalyzes C11α-hydroxylation of ent-cassadiene, and presumably mediates an early step in biosynthesis of the derived phytocassane class of phytoalexins. Here we report biochemical characterization of CYP76M5, -6, and -8. Our results indicate that CYP76M8 is a multifunctional/promiscuous hydroxylase, with CYP76M5 and -7 seeming to provide only redundant activity, while CYP76M6 seems to provide both redundant and novel activity, relative to CYP76M8. RNAi-mediated double knockdown of CYP76M7 and -8 suppresses elicitor inducible phytocassane production, indicating a role for these monooxygenases in phytocassane biosynthesis. In addition, our data suggests that CYP76M5, -6, and -8 may play redundant roles in production of the oryzalexin class of phytoalexins as well. Intriguingly, the preceding diterpene synthase for oryzalexin biosynthesis, unlike that for the phytocassanes, is not found in the chromosome 2 diterpenoid biosynthetic gene cluster. Accordingly, our results not only uncover a complex evolutionary history, but also further suggest some intriguing differences between plant biosynthetic gene clusters and the seemingly similar microbial operons. The implications for the underlying metabolic evolution of plants are then discussed.     

2'',3''=Carbonates in the synthesis of uridine 5''-deoxy and 2'',5''-dideoxy derivatives.

Detritylation of 2',3'-O-carbonyl-5'-O-trityluridine (Ia) with ethereal hydrogen chloride affords 2',3'-O-carbonyluridine (Ib; 83%) which is converted by mesylation to the 5'-mesylcarbonate Ic (75%). Reaction of compound, Ic with tetrabutylammonium bromide in DMF affords the 5'-bromo carbonate Id (77%) which is reduced with tributyltin hydride to the 5'-deoxyuridine 2',3'-cyclic carbonate Ie (70%). When heated with imidazole, compound Ie affords the 2,2'-anhydro derivative IIa (76%) which is converted to the 2'-chloro derivative IIIa (88%) on heating with HC1/DMF. The tributyltin hydride reduction of compound IIIa gives 2',5'-dideoxyuridine (IIIb; 68%). When heated with NaHCO3 in DMF, the 5'-bromo carbonate Id affords the anhydro bromo derivative IIb (50%) which is converted to the 2',5'-dichloro derivative IIIc (86%) on heating with HC1/DMF. The tributyltin hydride reduction of compound IIIc affords the 2',5'-dideoxy derivative IIIb (59%). Alkaline hydrolysis of the 2,2'-anhydro derivative IIa affords the arabinosyl derivative IVa which is converted to the diacetyl derivative IVb (34%) by acetylation. When refluxed in water, the 2',3'-cyclic carbonates Ib, Id, and Ie are hydrolysed to the parent nucleosides, namely, uridine (Va; 81%), 5'-bromo-5'-deoxyuridine (Vb; 78%), and 5'-deoxyuridine (Vc; 83%). Hydrolysis of carbonates Ib and Ie is accompanied by the formation of the 2,2'-anhydro derivatives IIc (10%) and IIa (5%) as by-products.     

Nucleotide sequence of the goat embryonic alpha globin gene (zeta) and linkage and evolutionary analysis of the complete alpha globin cluster

In previous studies we identified and sequenced clones containing two adult alpha globin genes of the goat. Additional studies have revealed the presence of an embryonic alpha globin gene termed zeta. Sequence analysis of the gene shows that it is the largest mammalian or avian globin gene cloned to date. Its unusual size is mainly due to a 14 base-pair tandem repeat sequence in its first intron. A similar sequence is also found in the first intron of the human zeta gene. The goat zeta coding sequence differs greatly from that of the adult alpha, particularly at amino acid position 38, where it codes for the amino acid replacement of Gln for Thr. This change may confer a higher intrinsic O2 affinity on the zeta globin protein, ensuring a sufficient O2 supply for the developing goat embryo. The cloning and sequencing of this gene completes the alpha globin locus of the goat, composed of three genes in the following order 5'-zeta-I alpha-II alpha-3'. Evolutionary comparisons of the goat alpha locus with other amphibian, avian and mammalian loci reveal several interesting features. Statistical analysis confirms the hypothesis that the embryonic alpha gene is much older (400 million years) than the embryonic beta gene (200 million years), and that it is descended from a primordial gene, whose present-day counterpart is the Xenopus larval alpha globin gene. Our results also suggest that after the divergence of the avian line, the alpha A gene converted the alpha D gene during the evolution of the pre-mammalian line. The alpha D globin gene remains unconverted in the avian line, potentially because of insertion/deletion sequences that may prevent any gene conversion event. The divergence rates of specific globin genes have been analyzed and found to form an essentially straight line, in agreement with the neutralist view of evolution.