Search

PipMaker (http://bio.cse.psu.edu) is a World-Wide Web site for comparing two long DNA sequences to identify conserved segments and for producing informative, high-resolution displays of the resulting alignments. One display is a percent identity plot (pip), which shows both the position in one sequence and the degree of similarity for each aligning segment between the two sequences in a compact and easily understandable form. Positions along the horizontal axis can be labeled with features such as exons of genes and repetitive elements, and colors can be used to clarify and enhance the display. The web site also provides a plot of the locations of those segments in both species (similar to a dot plot). PipMaker is appropriate for comparing genomic sequences from any two related species, although the types of information that can be inferred (e.g., protein-coding regions and cis-regulatory elements) depend on the level of conservation and the time and divergence rate since the separation of the species. Gene regulatory elements are often detectable as similar, noncoding sequences in species that diverged as much as 100-300 million years ago, such as humans and mice, Caenorhabditis elegans and C. briggsae, or Escherichia coli and Salmonella spp. PipMaker supports analysis of unfinished or "working draft" sequences by permitting one of the two sequences to be in unoriented and unordered contigs.

We have previously reported, on the basis of transfection experiments, the existence of a silencer element in the 5'-flanking region of the human embryonic (epsilon) globin gene, located at -270 base pairs 5' to the cap site, which provides negative regulation for this gene. Experiments in transgenic mice suggest the physiological importance of this epsilon-globin silencer, but also suggest that down-regulation of epsilon-globin gene expression may involve other negative elements flanking the epsilon-globin gene. We have now extended the analysis of epsilon-globin gene regulation to include the flanking region spanning up to 6 kilobase pairs 5' to the locus control region using reporter gene constructs with deletion mutations and transient transfection assays. We have identified and characterized other strong negative regulatory regions, as well as several positive regions that affect transcription activation. The negative regulatory regions at -3 kilobase pairs (epsilonNRA-I and epsilonNRA-II), flanked by a positive control element, has a strong effect on the epsilon-globin promoter both in erythroid K562 and nonerythroid HeLa cells and contains several binding sites for transcription factor GATA-1, as evidenced from DNA-protein binding assays. The GATA-1 sites within epsilonNRA-II are directly needed for negative control. Both epsilonNRA-I and epsilonNRA-II are active on a heterologous promoter and hence appear to act as transcription silencers. Another negative control region located at -1.7 kilobase pairs (epsilonNRB) does not exhibit general silencer activity as epsilonNRB does not affect transcription activity when used in conjunction with an epsilon-globin minimal promoter. The negative effect of epsilonNRB is erythroid specific, but not stage-specific as it can repress transcription activity in both K562 erythroid cells as well as in primary cultures of adult erythroid cells. Phylogenetic DNA sequence comparisons with other primate and other mammalian species show unusual degree of flanking sequence homology for the epsilon-globin gene, including in several of the regions identified in these functional and DNA-protein binding analyses, providing alternate evidence for their potential importance. We suggest that the down-regulation of epsilon-globin gene expression as development progresses involves complex, cooperative interactions of these negative regulatory elements, epsilonNRA-I/epsilonNRA-II, epsilonNRB, the epsilon-globin silencer and probably other negative and positive elements in the 5'-flanking region of the epsilon-globin gene.

The determination of long segments of DNA sequences encompassing the beta- and alpha-globin gene clusters has provided an unprecedented data base for analysis of genome evolution and regulation of gene clusters. A newly developed computer tool kit generates local alignments between such long sequences in a space-efficient manner, helps the user analyze the alignments effectively, and finds consistently aligning blocks of sequences in multiple pairwise comparisons. Such sequence analyses among the beta-like globin gene clusters of human, galago, rabbit, and mouse have revealed the general patterns of evolution of this gene cluster. Alignments in the flanking regions are very useful in assigning orthologous relationships. Investigation of such matches between the mouse and human beta-like globin gene clusters has led to a reassessment of some orthologous assignments in mouse and to a revision of the proposed pathway for evolution of this gene cluster. In general, the interspersed repetitive elements have inserted independently, presumably via a retrotransposition mechanism, in the different mammalian lineages. However, some examples of ancient L1 repeats are found, including one between the epsilon- and gamma-globin genes that appears to have been in the ancestral eutherian gene cluster. Prominent matching sequences are found in a long region 5' to the epsilon-globin gene, the locus control region (LCR) that is a positive regulator of the entire gene cluster. Three-way alignments among the human, goat, and rabbit sequences can extend for > or = 3 kb in part of the LCR (DNase hypersensitive site 3), indicating that the cis-acting components of this complex regulatory region cover a long segment of DNA. In contrast to the beta-like globin gene clusters, the alpha-like globin gene clusters of many mammals occur in very G+C-rich isochores and contain prominent CpG islands. The regions between the alpha-like globin genes are evolving faster than the intergenic regions of the beta-like globin gene clusters. The contrasts between the two gene clusters can be attributed to differences in DNA metabolism in the isochore. The proximal control elements of the rabbit alpha-globin gene are located both 5' to and within the gene. All of this region is part of a prominent CpG island that may be acting as an extended, enhancer-independent promoter. One can hypothesize that the analogue to the LCR in the alpha-globin gene cluster may interface with the distinctive alpha-globin promoter in ways different from the interaction between the beta LCR and the promoters of beta-like globin genes.(ABSTRACT TRUNCATED AT 400 WORDS)

The addition of two embryonic globin genes, zeta 0 and zeta 4, to the rabbit alpha-like globin gene cluster expands it to include eight genes arranged 5′-zeta 0-zeta 1-alpha 1-theta 1-zeta 2-zeta 3-theta 2-zeta 4-3′. The identification of these new genes supports the model that this gene cluster evolved by a series of block duplications of gene sets. The nucleotide sequence of three embryonic zeta-globin genes, zeta 1, zeta 2, and zeta 3, shows that all three are pseudogenes. Gene zeta 1 contains two frame-shift deletions, gene zeta 2 has lost exon 1 as well as the 5′ promoter sequences, and gene zeta 3 has lost and replaced codons for amino acids that are critical for the function of alpha- and zeta-globin polypeptides. However, genes zeta 1 and zeta 3 are still transcriptionally competent, as shown by the accurate initiation and processing of transcripts from cloned genes introduced into HeLa cells. A quantitative comparison of the zeta-globin gene sequences indicates that the ancestor to the rabbit zeta 1 and zeta 3 genes was inactivated about 44 million years ago, and the block duplication that formed the two genes occurred about 28 million years ago. About 600 base pairs of the 5′-flanking sequence of the rabbit zeta 3-globin gene is very similar to the 5′ flanks of three other mammalian zeta-globin genes.

The human beta-globin gene cluster is regulated in part by a distal locus control region that is required for opening a chromatin domain in erythroid cells and enhancing expression of the beta-like globin genes at the correct developmental stages. One part of the locus control region, called hypersensitive site 2 (HS2), functions as a strong enhancer. Matches to the consensus binding sites for basic helix-loop-helix (bHLH) proteins (E boxes) are well conserved within the HS2 core. We show that mutations of the HS2 core that alter an invariant E box cause a 3.5-fold reduction in enhancement of expression of an epsilon-globin reporter gene in transiently transfected K562 cells, both before and after induction. Mutations of the HS2 core that alter a less-highly conserved E box cause a more modest reduction in enhancement. Footprint analysis shows binding of erythroid nuclear proteins in vitro to the invariant E box as well as an adjacent CAC/GTG box. Probes containing the E box regions form sequence-specific complexes with proteins from both K562 and MEL nuclear extracts; these are disrupted by the same mutations that decrease enhancement. Some of these latter complexes contain known bHLH proteins, as revealed by specific loss of individual complexes when treated with antibodies against TAL1 and USF. Interaction between the E boxes and the bHLH proteins, as well as other binding proteins, could account for the role of these sites in enhancement by HS2.

The roles of each DNase hypersensitive site (HS), and the DNA sequences between them, in the activity of the locus control region of the mammalian beta-globin gene domain were examined by placing human and rabbit restriction fragments containing the cores of HS2, HS3, HS4, and HS5, along with varying amounts of flanking DNA, upstream of a hybrid epsilon-globin-luciferase reporter gene and testing for effects on expression both prior to and after integration into the chromosomes of K562 cells, a human erythroid cell line. Prior to integration, fragments containing HS2 enhanced expression to the greatest extent, and the modest enhancement by some fragments containing HS3 correlated with the presence of a well-conserved binding site for AP1/NFE2. The stronger effects of larger locus control region DNA fragments in clones of stably transfected cells indicates a role for sequences outside the HS cores after integration into the genome. The strong effect of a 1.9-kilobase HindIII fragment containing HS3 after, but not prior to, integration argues for the presence of a chromatin domain-opening activity. Use of a rabbit DNA fragment containing both HS2 and HS3 demonstrated a synergistic interaction between the two HSs when their natural context and spacing are preserved.

A cloned 13.3-kilobase (kb) region of rabbit genomic DNA contains a cluster of alpha-like globin genes arranged 5'-psi zeta-(3.6 kb)-alpha 1-(2.2 kb)-psi alpha-3'. Genomic blot hybridization data show that this is the major alpha-like globin gene cluster in rabbits, although a second alpha-globin gene (alpha 2) is also detected. Repetitive sequences from the C family of short repeats flank the psi zeta gene, and a new repetitive element, the F repeat is located 3' to psi alpha. The sequence was determined for a 4024-base pair (bp) segment that extends from 149 bp 5' to the cap site of alpha 1 to 207 bp 3' to psi alpha. Gene alpha 1 is functional and encodes one of the major allelic variants of rabbit alpha-globin. This gene has very short introns (77 bp in intron 1 and 83 bp in intron 2) and an unusual ATA box in the 5' flanking region (CTTAAA), which does function to promote transcription by RNA polymerase II in a cell-free system. Short (4 or 9 bp) G + C-rich repeats are interspersed throughout the flanking regions and the introns. Gene psi alpha cannot encode a globin polypeptide, and it is probably inactive. This is shown by the absence of a normal globin gene promoter, the replacement of the 5' untranslated sequence by tandem repeats of the sequence GCCCGCCGC, frameshift deletions in exon 2, and the modification of the polyadenylation signal to AGTAAA. The intergenic region between alpha 1 and psi alpha is very G + C rich (65.7% G + C) and contains many short, tandem repeats. Gene psi zeta hybridizes specifically to a human zeta-globin gene probe, but a partial sequence reveals frameshift mutations that probably make psi zeta a pseudogene. Mammalian alpha-globin gene clusters vary in the presence or absence of pseudogenes, and if present, the position of the pseudogene differs in various gene clusters. Among the mammalian alpha-like gene clusters so far analyzed, the rabbit gene cluster is unique in the absence of duplicated alpha-globin genes that are undergoing concerted evolution.

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.

We have hybridized pulse-labeled nuclear transcripts to cloned DNA fragments from the rabbit beta-like globin genes to determine the developmental timing, extent, and asymmetry of their transcription. The fetal-adult gene beta 1 was transcribed in fetal liver but not embryonic nuclei, whereas genes beta 3 and beta 4, which encode embryonic globin polypeptides, were transcribed only in embryonic nuclei. This shows that the switch from embryonic to fetal-adult globin production in rabbits is accomplished primarily by differential transcription of the beta-like globin genes. Gene beta 1 was subdivided into M13 subclones and tested for hybridization to nascent RNA. The nucleotide sequence of the 3' flanking region of gene beta 1 was also determined for 2,447 base pairs past the polyadenylation [poly(A)] site. No transcripts were found 5' to the cap site, but asymmetric transcription of gene beta 1 proceeded at a high level through the gene and past the poly(A) addition site for 603 nucleotides. The level of transcription declined after this, gradually dropping through the next 568 nucleotides. No polymerases were found on a fragment that begins 1,707 nucleotides past the poly(A) site; this fragment was part of a segment of repetitive DNA. These data show that the transcription unit of gene beta 1 begins at or near the cap nucleotide and extends at least 1,171 but no more than 1,706 nucleotides past the poly(A) addition site. The DNA segment that precedes the region of declining transcription contained an inverted repeat and encoded a short RNA transcribed by RNA polymerase II from the strand opposite the beta 1 transcript. These two features may function to attenuate the transcription of gene beta 1. An inverted repeat and a potential polymerase II transcription unit were also found in the homologous segment 3' to the human beta-globin gene. A short DNA segment close to the 3' end of the beta 1 transcription unit was transcribed more actively than the surrounding DNA, and it contained sequences that match the consensus internal control region for RNA polymerase III. This DNA segment may contain a separate polymerase III transcription unit. A member of the D repeat family located 3' to gene beta 1 was not transcribed in its entirety coordinately with beta 1.

In the rabbit, two beta-like globin genes, beta 3 and beta 4, are expressed in embryonic erythrocytes and one, beta 1, is expressed in fetal and adult erythrocytes. The complete nucleotide sequence of embryonic gene beta 4 is presented, including the region from 582 base pairs before the cap site to 508 base pairs after the poly(A) addition site. beta 4 has the tripartite structure characteristic of animal globin genes, being interrupted by intervening sequences within codon 30 and between codons 104 and 105. Conserved sequences thought to play roles in transcription, RNA processing, and translation are found in beta 4. Rabbit gene beta 4 shows substantial homology to the human embryonic epsilon-globin gene, not only in message-coding blocks, but also in the 5' and 3' flanking regions and in the two intervening sequences. Comparison of all four rabbit beta-like genes with those of humans suggests that a primordial four gene family, 5'-epsilon-gamma-delta-beta-3', existed prior to the mammalian radiation. An alignment of the 5' flanking sequences of a collection of animal beta-like globin genes shows some conserved regions that differ between the adult genes and the embryonic/fetal genes. These could reflect a common evolutionary lineage rather than stage-specific control sequences.

Despite their descent from a common ancestral gene and the requirement for coordinated, tissue-specific regulation, the alpha- and beta-globin genes in many mammals are regulated in distinctly different ways. Unlike the beta-globin gene, the rabbit alpha-globin gene is transiently expressed at a high level without an added enhancer in transfected erythroid and non-erythroid cells. By examining a series of alpha/beta fusion genes, we show that internal sequences of the rabbit alpha-globin gene (within the first two exons and introns) are required along with the 5' flank for this enhancer-independent expression. Furthermore, deletion of the introns of the alpha-globin gene, or replacement by introns of the beta-globin gene, results in severely decreased expression of the transfecting genes. Hybrid constructs between segments of the alpha-globin gene and a luciferase gene confirm that internal alpha-globin sequences are needed for high level production of RNA in transfected cells. The flanking and internal sequences implicated in regulation of the rabbit alpha-globin gene coincide with a prominent CpG-rich island and may comprise an extended promoter (including both flanking and intragenic sequences) that is active in transfected cells without an enhancer.

C repeats constitute the predominant family of short interspersed repeats (SINEs) in the rabbit genome. Determination of the nucleotide sequence 5' to rabbit zeta-globin genes reveals clusters of C repeats, and analysis of these and other sequenced regions of rabbit chromosomes shows that the C repeats have a strong tendency to insert within or in close proximity to other C repeats. An alignment of 44 members of the C repeat family shows that they are composites of different sequences, including a tRNA-like sequence, a conserved central core, a stretch of repeating CT dinucleotides, and an A-rich tract. Cladograms generated by both parsimony and cluster analysis subdivide the C repeats into at least three distinct subfamilies. Nucleotides at sites diagnostic for subfamilies appear to have changed in a punctuated and progressive manner during evolution, indicating that a limited number of progenitors have given rise to new repeats in waves of dispersion. C repeats that insert into preexisting C repeats belong to subfamilies that are proposed to have been propagated more recently; hence, these data support the model of dispersion in successive waves. The divergence among the oldest group of C repeats is greater than that observed for the analogous Alu repeats in humans, indicating that rabbit C repeats have been propagating longer than human Alu repeats. The improved consensus sequence for these repeats is similar to that of the predominant artiodactyl SINE in both the tRNA-like region and a central region. Because members of different subfamilies cross-hybridize very poorly, hybridization data with representatives of each subfamily provide a new minimal estimate, 234,000, for the copy number of C repeats in the rabbit haploid genome, although it is likely that the actual value is closer to 1 million.

Analysis of 37 short repetitive elements (SINEs) in rabbit DNA that are known as C repeats has revealed three that contribute functional polyadenylation signals to genes into which they have been inserted. Similar roles have been attributed to particular individual SINEs in rodents and primates before, suggesting that these roles may be common to SINEs in all mammalian orders. Although most SINEs appear to have little influence on the genome individually, the observation that three of 36 rabbit C repeats provide functional sequences suggests a mechanism for the maintenance of SINEs within mammalian genomes.

The alpha-like globin gene cluster in rabbits contains embryonic zeta-globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1-zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1-zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.