Repetitive DNA sequences near three human beta-type globin genes.

Five repetitive DNA sequences, of average length 259 bp, have been identified in the intergenic regions which flank three human beta-tupe globin genes. A pair of inverted repeat sequences, separated by 919 bp, was found 1.0 kb to the 5' side of the epsiln-globin gene. Each contains a homologous Alu I site. Another repetitive sequence, with the same orientation as the inverted repeat sequence closest to the epsilon-globin gene, lies about 2.2 kb to the 5' side of the delta-globin gene. A pair of inverted repeat sequences, with the same relative orientations as the other pair and separated by about 800 bp, was found about 1.5 kb to the 3' side of the beta-globin gene.     

Highly variable regions of DNA flank the human alpha globin genes.

A series of restriction fragment length polymorphisms which are due to DNA rearrangements have been identified within two highly variable regions flanking the human alpha globin genes. The existence of such highly polymorphic areas provides a large number of individual genetic markers for the alpha globin gene cluster on chromosome 16. If, as seems likely, such regions occur frequently throughout the human genome they should be of considerable value in the antenatal diagnosis of genetic disease.     

DNA sequences flanking the starts of the hsp 70 and alpha beta heat shock genes are homologous

To determine whether ribosomes have a role in the postfertilization activation of protein synthesis in sea urchin eggs, we measured the translational activity of ribosomes isolated from unfertilized eggs and embryos of Strongylocentrotus purpuratus. Numerous previous studies have indicated few if any differences in the activity of such ribosomes. However, by using improved physiological isolation and in vitro conditions, we have found important differences in the activities of egg and embryo ribosomes. Ribosomes obtained from blastula polyribosomes were active in translating reticulocyte mRNA in a ribosome-dependent cell-free translation system, whereas ribosomes obtained from unfertilized eggs became fully active only after a characteristic, reproducible delay of up to 15 min at 26°C. The extent of this delay varied with incubation pH, but not with concentrations of K⁺, Mg²⁺, initiation factors, or mRNA. However, at incubation pH between 6.90 and 7.65, the egg ribosomes were always less active than blastula ribosomes.     

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.     

Genomic organization and DNA sequences of human 17 beta-hydroxysteroid dehydrogenase genes and flanking regions. Localization of multiple Alu sequences and putative cis-acting elements.

Genomic 17 beta-hydroxysteroid-dehydrogenase (17-HSD) clones were isolated from a human leucocyte genomic library using cDNA encoding human placental 17-HSD as a probe. The overlapping fragments spanned more than 21 kbp containing the duplications, 6.2 kbp of each, as well as 7 kbp upstream and 1.6 kbp downstream from the duplicated sequences. 17 complete and eight partial Alu elements were clustered in this area, covering about 30% of the region, including the borders of the duplications. Each duplication contained a 17-HSD gene and a conserved region of 1.56 kbp with 98% intercopy similarity. The exon structure of the 17-HSD gene II corresponded to the known cDNA species, but both genes contained a possible promoter region with TATA, GC and inverse CAAT boxes. The 5' flanking regions contained sequences similar to the consensus sequences of cis-acting elements, defined as regulators of 17-HSD gene expression. These putative sequences included estrogen and progesterone/glucocorticoid-response elements and a cyclic-AMP regulatory element.     

DNA methylation: sequences flanking C-G pairs modulate the specificity of the human DNA methylase.

Synthetic single-stranded oligodeoxynucleotides of known sequence have been used as in vitro substrates for a partially purified HeLa cell DNA methylase. Although most oligonucleotides tested cannot be used by the HeLa DNA methylase in vitro, we have found a unique 27mer, containing 2 C-G pairs, that is an excellent substrate for the enzyme. Analysis of the methylation of the 27mer, its derivatives and other oligomer substrates reveal that the HeLa DNA methylase does not significantly methylate an oligomer which contains just one C-G pair. In addition, only one of the two C-G pairs in the 27mer is methylated and this methylation is abolished if the other C-G pair is converted to a C-A pair. Furthermore, the HeLa enzyme apparently cannot methylate C-G pairs located in compounds containing a high A + T content. The most efficient methylation occurs with multiple separated C-G pairs in a compound with a high G + C content (greater than 65%). The results suggest that clustering of C-G pairs in regions of the DNA high in G + C content may be the preferred site for DNA methylation in vivo.     

Intervening sequences in human fetal globin genes adopt left-handed Z helices

The large intervening sequences ( IVS2 ) of three human fetal globin genes contain tracts of alternating purine-pyrimidine sequences approximately 40-60 base pairs in length which adopt left-handed Z DNA helices under the influence of negative supercoiling. The amount of negative supercoiling (approximately 0.045) required for the right- to left-handed transitions is within the physiological range. The structural aberrations between the right- and left-handed helices were mapped by sequencing the S1 nuclease cleavage sites. Two-dimensional gel electrophoretic analyses of the supercoil-induced relaxation served to characterize the type and length of left-handed structure. Furthermore, binding studies with several types of antibodies confirmed the presence of left-handed helices. Since these simple sequences appear to be hotspots for recombination and gene conversion, unusual DNA conformations may participate in genetic expression.     

A short repeat in the genome of the DNA sequences flanking bovine growth hormone genes

The phage clones containing a gene coding for bovine growth hormone were isolated from a bovine genomic library. Comparison of the 5' and 3' regions flanking the bovine growth hormone gene by Southern blot hybridization revealed that they share homology. Screening the bovine genomic library by nick-translated DNA fragment from 5' flanking region leads to conclusion that this sequence is present in 0.1% of clones. Each analysed clone carrying the sequence contains some copies of it.     

Restriction endonuclease analysis of human globin genes in cellular DNA

Using samples of human cellular DNA digested with restriction endonucleases Eco RI, Hind III, Hinc II, Bam HI, Alu I, or Hae III, we were able to localize globin gene fragments separated by agarose gel electrophoresis. The fragments were transferred to nitro-cellulose filters and identified by hybridization to [³²P] cDNA for total adult globin mRNA. The α-globin gene fragments were specifically identified by their presence in normal controls and absence in DNA from homozygous α-thalassemia, a genetic disorder due to deletion of α-globin genes. In addition, the patterns with Hind III indicate a 4.1 kb distance between the centers of the normal duplicated α-globin gene loci.     

A chimpanzee-derived chromosome-specific alpha satellite DNA sequence conserved between chimpanzee and human

We describe a cloned 2.7 kb alpha satellite sequence, Pan-3, from the pygmy chimpanzee (Pan paniscus) that specifically hybridizes in situ to chromosome 19 in the pygmy chimpanzee and to the homeologous human chromosome, no. 17. Using high stringency conditions of hybridization on Southern blots, this sequence hybridized to DNA from both species of chimpanzee (P. paniscus and P. troglodytes) and from human but not to DNA from gorilla (Gorilla gorilla) or orangutan (Pongo pygmaeus). Partial sequence analysis showed that Pan-3 and a previously described human chromosome 17-specific clone have up to 91% sequence identity. To our knowledge this is the highest sequence similarity reported between alphoid subsets from human and any other primate.by T.C. Hsu     

Cloned embryonic DNA sequences flanking the mouse immunoglobulin C gamma 3 and C gamma 1 genes

To investigate the DNA surrounding genes for immunoglobulin heavy chain constant (CH) regions, we have isolated two clones bearing a C gamma 3 gene and two bearing a C gamma 1 gene from a library of mouse embryo DNA fragments. The C gamma 3 clones span 8.6 kilobase pairs (kb) on the 5' side of the gene and 6.7 kb on its 3' side, while the C gamma 1 clones together span 13 kb of 5' flanking sequence and 2.5 kb of 3' flanking sequence. Restriction mapping of the C gamma 3 gene indicates that intervening sequences divide the gene into segments of domain size, as in other CH genes. Hybridization of clone fragments to restriction digests of mouse DNA indicates that both the C gamma 1 and C gamma 3 genes probably occur as single copies in the genome. Moreover, the entire cloned sequences on the 5' side of both genes appear to be unique in the genome, indicating that no large common sequences flank CH genes. Restriction data suggest that the C gamma 3 gene is 37-40 kb 5' to the C gamma 1 gene.     

Human fetal globin DNA sequences suggest novel conversion event.

DNA sequencing studies of two recently cloned human A gamma globin alleles has revealed a number of base differences which are clustered in the large intron (IVS-2). One allele has a previously undescribed IVS-2 sequence. Most of the allelic differences can be explained as resulting from a gene conversion event involving G gamma as a donor. A novel feature of this event is that three G gamma-like regions occur interspersed among unconverted areas of the A gamma gene. We propose that an alternating purine-pyrimidine run which is located between two of the converted sites is the initiation site of the conversion event. Consistent with models of gene conversion, this poly (purine-pyrimidine) tract has single-stranded characteristics in supercoiled plasmids as assayed by S1-nuclease.     

Transfer RNA genes in Drosophila mitochondrial DNA: related 5'' flanking sequences and comparisons to mammalian mitochondrial tRNA genes.

Genes for tRNAgly and tRNAserUCN have been identified within sequences of mtDNA of Drosophila yakuba. The tRNAgly gene lies between the genes for cytochrome c oxidase subunit III and URF3, and all three of these genes are contained in the same strand of the mtDNA molecule. The tRNAserUCN gene is adjacent to the URF1 gene. These genes are contained in opposite strands of the mtDNA molecule and their 3' ends overlap. The structures of the tRNAgly and tRNAserUCN genes, and of the four tRNA genes of D. yakuba mtDNA reported earlier (tRNAile, tRNAgln, tRNAf-met and tRNAval) are compared to each other, to non-organelle tRNAs, and to corresponding mammalian mitochondrial tRNA genes. Within 19 nucleotides upstream from the 5' terminal nucleotide of each of the Drosophila mitochondrial tRNAgly, tRNAserUCN, tRNAile, tRNAgln and tRNAf-met genes occurs the sequence 5'TTTATTAT, or a sequence differing from it by one nucleotide substitution. Upstream from this octanucleotide sequence, and separated from it by 3, 4 and 11 nucleotides, respectively, in the 5' flanking regions of the tRNAile, tRNAserUCN and tRNAgly genes occurs the sequence 5'GATGAG.     

Molecular cloning of a family of retroviral sequences found in chimpanzee but not human DNA.

A number of retrovirus-like sequences have been cloned from chimpanzee DNA which constitute the chimpanzee homologs of the endogenous colobus type C virus CPC-1. One of the clones contains a nearly complete viral genome, but others have sustained deletions of 1 to 2 kilobases in the polymerase gene. The pattern of related sequences detected in other primate species is consistent with the genetic transmission of these sequences for millions of years. However, the appropriately related sequences have not been detected in human, gibbon, or orangutan DNAs. These results suggest either that this family of sequences has been deleted from humans, gibbons, and orangutans, or that the genes were recently acquired in the chimpanzee and gorilla lineages.     

The divergence between human and baboon globin genes.

Complementary DNA (cDNA) was prepared with viral RNA-directed DNA polymerase from purified baboon globin messenger RNA (mRNA). Homologous and heterologous hybrids between human and baboon mRNAs and cDNAs were compared for extent of hybridisation and thermal stability. Higher mRNA inputs to the hybridizations were required to reach saturation in the heterologous cases. The melting temperature of the heterologous hybrid was 5 degrees C lower than the homologous hybrid. Between these two primates, divergence has occurred in the globin gene to a smaller extent than that possible from third position changes in the coding sequences of the divergence of total DNA. Globin cDNA prepared from baboon will not in general be useful as a probe for human globin mRNA or human globin gene sequences.