Transposition of a long member of the L1 major interspersed DNA family into the mouse beta globin gene locus.

A long member of the highly repeated long interspersed DNA family L1Md (for L1 in Mus domesticus) has integrated by transposition into a target site which lies between the two adult beta globin genes of mouse. DNA hybridization and nucleotide sequence analysis show that this target site, which is part of the single copy DNA flanking the globin genes, is interrupted by the L1 element in one chromosome but is uninterrupted in both allelic and ancestral chromosomes. Other large DNA rearrangements of the region between the two adult beta globin genes are also associated with these allelic chromosomes, and include insertions or deletions of both single copy DNA and simple and complex repetitive DNA. This has caused extensive reorganization of this intergenic region. However, the distance between the two genes flanking this region remains conserved, suggesting that the spacing of the globin genes may be subject to conservative selection.     

A composite transposon 3'' to the cow fetal globin gene binds a sequence specific factor.

Two unusual sequence organizations were found within the beta-globin locus of the cow. Each was a composite, consisting of closely linked Alu-type repeats with a short stretch of genomic non-repetitive sequence, called a lagan, sandwiched between. One lagan was found 3' to the fetal globin gene, while the second lay between the adult globin gene and a globin pseudogene. Southern blot analysis indicated that both lagans appeared twice within the cow haploid genome, with the second copies lying outside the cow beta-globin locus. One of these non-globin locus homologues was cloned and subjected to sequence analysis. Comparison of the DNA sequence data showed that the lagan-Alu composite was transposed as a unit. The lagan 3' to the cow fetal globin gene contains the recognition site for a sequence specific DNA binding factor. This factor was present in extracts from fetal, but not from adult cow tissues.     

The evolution of a plant globin gene family

Summary We have analyzed the sequences of soybean leghemoglobin genes as an initial step toward understanding their mode of evolution. Alignment of the sequences of plant globin genes with those of animals reveals that (i) based on the proportion of nucleotide substitutions that have occurred at the first, second, and third codon positions, the time of divergence of plant and animal globin gene families appears to be extremely remote (between 900 million and 1.4 billion years ago, if one assumes constancy of evolutionary rate in both the plant and animal lineages) and (ii) in addition to the normal regulatory sequences on the 5 end, an approximately 30-base-pair sequence, specific to globin genes, that surrounds the cap site is conserved between the plant and animal globin genes. Comparison of the leghemoglobin sequences with one another shows that (i) the relative amount of sequence divergence in various coding and noncoding regions is roughly similar to that found for animal globin genes and (ii) as in animal globin genes, the positions of insertions and deletions in the intervening sequences often coincide with the locations of direct repeats. Thus, the mode of evolution of the plant globin genes appears to resemble, in many ways, that of their animal counterparts. We contrast the overall intergenic organization of the plant globin genes with that of animal genes, and discuss the possibility of the concerted evolution of the leghemoglobin genes.     

Conserved sequences and cell-specific DNase I hypersensitive sites upstream from the co-ordinately expressed alpha I- and alpha II-globin genes of Xenopus laevis

The globin gene family of Xenopus laevis comprises pairs of closely related genes that are arranged in two clusters, each pair of genes being co-ordinately and stage-specifically expressed. To get information on putative regulatory elements, we compared the DNA sequences and the chromatin conformation 5' to the co-ordinately expressed adult alpha-globin genes. Sequence analysis revealed a relatively conserved region from the cap site up to position -289, and further upstream seven distinct boxes of homology, separated by more diverged sequences or deletions/insertions. The homology boxes comprise 22 to 194 base-pairs showing 78 to 95% homology. Analysis of chromatin conformation showed that DNase I preferentially cuts the upstream region of both genes at similar positions, 5' to the T-A-T-A and the C-C-A-A-T boxes, only in chromatin of adult erythroblasts and erythrocytes, where adult globin genes are expressed, but not in chromatin of adult liver cells or larval erythrocytes, where these genes are silent. This suggests that cell- and stage-specific activation of these genes coincides with specific changes in chromatin conformation within the proximal upstream region. No difference was found in the nucleotide sequence within the DNase I hypersensitive region proximal to the adult alpha 1-globin gene in DNA from embryonic cells, in which this gene is inactive, and adult erythrocytes, expressing this gene.     

Conservation and variation in the large scale organisation of the globin gene domains of duck and chicken

Summary The genomic DNA of cloned recombinants containing the duck globin genes was compared to that of the analogous domains of the chicken. A 36 kb insert including the three alpha-type globin genes was isolated from a newly prepared duck genomic library in the cosmid PJB8; another recombinant contained a 45 kb insert with the four beta globin genes. In the alpha globin gene domain, the relative positions of genes, of repetitive sequences, and of the A+T-rich segments (AT-rich linkers, ATRLs) which frame the gene cluster (Moreau et al. 1982), were found to be closely maintained between duck and chicken. Although ATRLs and repetitive sequences also frame the gene cluster in the beta globin domains of duck and chicken, there is more genetic drift in their relative positions than in the alpha domain. It is of interest that several repetitive DNA segments were detected in the chicken beta globin domain which do not exist in corresponding positions in the duck. In view of the strict conservation in both species of genes and their relative positions in the cluster, this observation seems to exclude a simple function of repetitive sequences in the control of individual genes. The data are discussed with regard to the possible significance of repetitive and AT-rich DNA segments in genome organisation and function.     

A physiological delay in human fetal hemoglobin switching is associated with specific globin DNA hypomethylation

The human fetal-to-adult globin switch normally occurs on a fixed schedule, beginning at 32-34 weeks gestation, and recent studies have suggested an association between this developmental inactivation of the fetal (gamma) globin genes and the appearance of methylation within and around these genes. We have studied a population of infants in whom this switch does not occur before birth (infants of diabetic mothers, IDM) and examined the patterns of methylation surrounding their active gamma-globin genes, in comparison to the gamma-globin genes of age-matched controls who have switched their pattern of globin gene expression on schedule. All genomic DNA samples from infants with delays in the globin switch demonstrated extensive hypomethylation in the region of the gamma-globin genes, comparable to that found in the genomes of fetuses of less than 21 weeks gestation. DNA from the erythroid cells of infants of 32-40 weeks gestation had no detectable hypomethylation in the gamma-globin region. These findings support the concept that hypomethylation is an accurate developmental marker of globin gene switching, and suggest that globin gene expression in IDM may be arrested at an early preswitch stage.     

The isolation of the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene from a goat DNA recombinant library

As an approach to understand how the expression of globin genes are regulated during development, clones containing globin DNA sequences were selected from a recombinant library of goat genomic DNA. The type of globin gene present in each of the recombinants was determined by cross-hybridization to the DNA of mouse alpha- and beta-globin cDNA-containing plasmids. Of 11 clones isolated, eight hybridized specifically to the DNA of the mouse beta-globin plasmid, while one clone hybridized only to the DNA of the alpha globin plasmid. The location of each globin sequence within its DNA insert was determined by a combination of restriction enzyme mapping and Southern transfer-hybridizations. Selected fragments were sequenced; comparisons of the amino acids coded for by these regions with those of the goat globins identified clones carrying beta A-, beta C-, and gamma-globin genes. Another recombinant coded for amino acid sequences resembling, but not identical with, the known goat globins, and was identified tentatively as containing an embryonic or epsilon-gene. Detailed analysis of the clone containing the beta C gene and an overlapping clone revealed that three other beta-like sequences are located 6, 12, and 21 kilobases on the 5'-side of the beta C gene. The globin sequence of the locus nearest to the beta C gene has an altered translation termination codon and, if transcribed and translated, would give a globin chain seven amino acids longer than the normal goat beta C-globin. In addition, the sequence following this termination codon is very AT-rich, unlike that of other globin genes. The recombinants described contain extensive regions of DNA surrounding the globin genes, making them useful for identifying regulatory sequences as well as determining the sequence organization of the goat globin genes.     

DNA methylation in the human γδβ-globin locus in erythroid and nonerythroid tissues

We have analyzed DNA modification in the human γδβ-globin gene region at 17 cleavage sites of restriction endonucleases which are unable to cleave DNA if 5-methylcytosine is present at certain positions in their respective cleavage sites. Using this criterion, all sites tested in the globin gene region are fully modified in the germ line (sperm) DNA. In somatic tissues, however, methyl groups are absent at specific sites in the globin gene region. In tissues not expressing the genes, these losses range from one of these cleavage sites in lymphocyte DNA to essentially all of these sites in the entire region in placental DNA. In the DNA of tissues expressing the globin genes, the region surrounding and including the genes expressed shows a low level of modification, whereas the neighboring DNA regions have a high level of modification. The data suggest that a low level of DNA methylation may be a necessary, but not a sufficient, condition for gene expression in higher eucaryotes.     

Study of markers of human erythroid differentiation in hybrid cells.

Somatic cell hybrids exhibiting co-expression of the globin genes of two species were generated by fusion of mouse erythroleukemia cells with Chinese hamster or human marrow erythroid cells. In contrast, extinction of the mouse globin genes occurred in hybrids formed between the erythroleukemia cells and human fibroblasts. Direct detection of the human globin genes in human X mouse fibroblast hybrids was achieved by annealing of DNA from these cells to human globin complementary DNA. This method was developed to permit the chromosomal assignment of the human globin genes.     

DNA sequences regulating human beta globin gene expression.

Human delta globin is expressed at approximately 1-2% of the level of human beta globin in erythroid cells despite the marked homology between these two globins. To determine the DNA sequences responsible for this effect, delta and beta globin genes and fusion products of these genes constructed in vitro were transfected and expressed in HeLa cells. The results indicate that when the small intervening sequence of the beta gene (beta IVS 1) is replaced by delta IVS 1, expression of the chimeric gene is the same as that of the normal beta globin gene. By contrast, when the large intervening sequence of the beta gene (beta IVS 2) is replaced by delta IVS 2, expression of the chimeric gene is markedly reduced. These results suggest that there are signals within IVS 2 of the delta and beta genes which affect their relative expression.     

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.     

Clonal Expansion Analysis of Transposon Insertions by High-Throughput Sequencing Identifies Candidate Cancer Genes in a PiggyBac Mutagenesis Screen

Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10⁶ reads corresponding to >10⁴ insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors.     

Gene reorganization during serial divisions of normal human cells

We have followed during serial divisions of human fibroblasts the presence in chromosomal and extrachromosomal DNA, of two genes that are expressed in fibroblasts, actin and interferon, and of one that is not expressed, globin. The intensity of the blot hybridization of the actin and globin probes with chromosomal DNA diminished during serial divisions of diploid fibroblasts. The interferon gene remained constant throughout the human fibroblast life span. Chromosomal DNA sequences were present in extrachromosomal circular DNA which appeared at the end of the fibroblast life span. The results could explain some functional changes that occur in these cell populations when their division potential declines.