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 structure of the Adh locus of Drosophila mettleri: an intermediate in the evolution of the Adh locus in the repleta group of Drosophila.

Members of species of the mulleri and hydei subgroups of the repleta group of Drosophila have duplicate Adh genes. The Adh regions of D. mojavensis, D. mulleri, and D. hydei contain three genes--a pseudogene, Adh-2, and Adh-1--arranged 5' to 3'. To understand the evolution of the triplicate Adh structure, we have cloned and sequenced the Adh locus of D. mettleri. This region consists of a 5' pseudogene and a 3' functional Adh gene. On the basis of the structure and nucleotide sequence comparisons of Adh genes of D. mettleri and other species, we propose that an initial duplication of the ancestral Adh gene generated two Adh genes arranged in tandem. The more 5' Adh gene became a pseudogene, while the more 3' gene remained functional through all the developmental stages. A second duplication of this 3' gene resulted in Adh regions with three genes--a pseudogene, Adh-2, and Adh-1.     

An expressed beta-tubulin gene, TUBB, is located on the short arm of human chromosome 6 and two related sequences are dispersed on chromosomes 8 and 13

The chromosomal assignments of an expressed beta-tubulin gene and two related sequences have been determined by Southern blot analysis of DNA from a panel of human x Chinese hamster somatic cell hybrids cleaved with Hind III or EcoRI. Probes containing the 3' untranslated regions of the expressed gene M40 and of pseudogene 21 beta were used to localize the M40 sequence (gene symbol TUBB) to chromosome 6 region 6p21----6pter, the 21 beta pseudogene (TUBBP1) to chromosome 8 region 8q21----8pter and a third related sequence (TUBBP2) to chromosome 13. Asynteny of expressed genes and related processed pseudogenes has now been demonstrated for several gene families.     

Cloning and expression of a beta tubulin gene of Physarum polycephalum

A beta tubulin gene of Physarum polycephalum has been isolated from a genomic library in the phage EMBL4. Southern-blot hybridization to genomic DNA indicates that the cloned DNA is derived from the betB1 locus of the beta tubulin gene family. A tubulin-specific subfragment of the phage DNA was used as a hybridization probe to construct a restriction map of the betB1 locus. The probe consisted of the almost complete coding region of the 5' half of the tubulin gene, interrupted by one intron. The derived amino acid sequence of this subclone deviates from the protein sequence for Physarum amoebal beta tubulin (amino acids 4-207) in two of 207 amino acids. We used both recA and recBC sbcB bacterial host strains, which have been recommended for cloning of instability-conferring sequences of the Physarum genome, but were unable to subclone the 3' part of the gene from the phage DNA. Primer-extension analysis indicates that the betB gene is expressed in the vegetatively proliferating amoebal and plasmodial stages of the life cycle as well as in differentiating (sporulating) plasmodia.     

Cloning of the genomic sequence encoding a processed adenylate kinase 2 pseudogene

A chromosomal DNA sequence harboring a processed AK2B pseudogene was isolated from a human genomic library. It was a variant of the AK2B gene sequence including several point mutations, deletions, and insertions. The nucleotide sequence of the ORF of the AK2B pseudogene predicted a truncated form of the AK2B mutant suggesting that the processed pseudogene is nonfunctional. A repetitive sequence, AAAAGAGAG, found in the 5' and 3' flanking regions of the pseudogene and the poly(A) tract in the 3' end junction suggest that a mRNA of AK2B may have been converted to the processed pseudogene by retrotransposition events. Previously, it was suggested that an adenylate kinase (AK) 2 related gene on chromosome 2, confirmed by Southern analysis using somatic cell hybrid cell lines, may be a processed pseudogene. It is proposed that the processed pseudogene isolated in this study may be the AK2 related nonfunctional gene localized on human chromosomes 2.     

Characterization of the human laminin beta2 chain locus (LAMB2): linkage to a gene containing a nonprocessed, transcribed LAMB2-like pseudogene (LAMB2L) and to the gene encoding glutaminyl tRNA synthetase (QARS).

The laminin beta2 chain is an important constituent of certain kidney and muscle basement membranes. We have generated a detailed physical map of a 110-kb genomic DNA segment surrounding the human laminin beta2 chain gene (LAMB2) on chromosome 3p21.3-->p21.2, a region paralogous with the chromosome 7q22-->q31 region that contains the laminin beta1 chain gene locus (LAMB1). Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified. The 3' end of LAMB2 lies 16 kb from the 5' end of the glutaminyl tRNA synthetase gene (QARS). About 20 kb upstream of LAMB2 we found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L). The sequence of 1.75 kb of genomic DNA at the 3' end of LAMB2L was similar to exons 8-12 of the laminin beta2 chain gene. The LAMB2L-LAMB2-QARS cluster lies telomeric to the gene encoding the laminin-binding protein dystroglycan (DAG1).     

Cloning of a Drosophila melanogaster adenine phosphoribosyltransferase structural gene and deduced amino acid sequence of the enzyme

The Aprt locus of Drosophila melanogaster encodes the structural gene for adenine phosphoribosyltransferase (APRT). DNA cloned from microdissected salivary gland polytene chromosome region 62B7-12 was used in conjunction with chromosome walking and hybrid selection of mRNA to isolate the Aprt gene. Aprt lies at cytogenetic position 62B9 and is closely flanked by other genes of unknown function. Nucleotide sequencing shows that four APRT cDNAs have a common 5' terminus with an apparent cap consensus sequence but two different 3' sites of polyadenylation. The distribution of conserved amino acid sequences in APRT from vertebrates, insects and bacteria suggests that they may have shared a common ancestral gene for this ubiquitous enzyme.     

Regulation of the replication of the murine immunoglobulin heavy chain gene locus: evaluation of the role of the 3' regulatory region.

DNA replication in mammalian cells is a precisely controlled physical and temporal process, likely involving cis-acting elements that control the region(s) from which replication initiates. In B cells, previous studies showed replication timing to be early throughout the immunoglobulin heavy chain (Igh) locus. The implication from replication timing studies in the B-cell line MPC11 was that early replication of the Igh locus was regulated by sequences downstream of the C alpha gene. A potential candidate for these replication control sequences was the 3' regulatory region of the Igh locus. Our results demonstrate, however, that the Igh locus maintains early replication in a B-cell line in which the 3' regulatory region has been deleted from one allele, thus indicating that replication timing of the locus is independent of this region. In non-B cells (murine erythroleukemia cells [MEL]), previous studies of segments within the mouse Igh locus demonstrated that DNA replication likely initiated downstream of the Igh gene cluster. Here we use recently cloned DNA to demonstrate that segments located sequentially downstream of the Igh 3' regulatory region continue to replicate progressively earlier in S phase in MEL. Furthermore, analysis by two-dimensional gel electrophoresis indicates that replication forks proceed exclusively in the 3'-to-5' direction through the region 3' of the Igh locus. Extrapolation from these data predicts that initiation of DNA replication occurs in MEL at one or more sites within a 90-kb interval located between 40 and 130 kb downstream of the 3' regulatory region.     

Chromosomal arrangement of leghemoglobin genes in soybean.

A cluster of four different leghemoglobin (Lb) genes was isolated from AluI-HaeIII and EcoRI genomic libraries of soybean in a set of overlapping clones which together include 45 kilobases (kb) of contiguous DNA. These four genes, including a pseudogene, are present in the same orientation and are arranged in the order: 5'-Lba-Lbc1-Lb psi-Lbc3-3'. The intergenic regions average 2.5 kb. In addition to this main Lb locus, there are other Lb genes which do not appear to be contiguous to this locus. A sequence probably common to the 3' region of Lb loci was found flanking the Lbc3 gene. The 3' flanking region of the main Lb locus also contains a sequence that appears to be expressed more abundantly in root tissue. Another sequence which is primarily expressed in root and leaf is found 5' to two Lb loci. Overall, the main leghemoglobin locus is similar in structure to the mammalian globin gene loci.     

Chromosome mapping and phylogenetic analysis of the cytosolic acetyl-CoA carboxylase loci in wheat

The cytosolic isoform of plant acetyl-CoA carboxylase is a multidomain enzyme involved in the synthesis of very-long-chain fatty acids and in secondary metabolism. Chromosome mapping of wheat identified one locus containing cytosolic acetyl-CoA carboxylase genes (Acc-2) and a related partially processed pseudogene (Psi-Acc-2) in the distal region of the long arm of wheat homoeologous group 3 chromosomes. Multiple copies of the Acc-2 genes, whose presence was suggested by sequence analysis, are likely to be arranged in tandem repeats. At least three out of five genes cloned from hexaploid wheat map to this locus. Another locus containing Acc-2--related sequences is present in the distal region of the long arm of chromosome 5D. The identity of the hybridizing DNA present at this locus remains unknown. A system based on PCR-cloning and DNA sequence analysis of acetyl-CoA carboxylase genes was developed to address various phylogenetic and systematics questions in grasses. It was applied to reconstruct the phylogeny of the Acc-2 genes from D- and S-genome Aegilops and A-genome Triticum diploid species, AABB- and AAGG-genome tetraploid wheat, and AABBDD-genome hexaploid wheat, as well as from rye and barley. The combined cytogenetic and molecular evolution approach allowed assignment of gene sequences included in phylogenetic analysis to specific loci on homoeologous chromosomes. Recurring gene duplication followed by chromosome translocation and/or possible loss of some gene copies, as well as loss of introns, occurred in the gene family in different plant lineages. Two major Acc-2 clades appeared before the divergence of barley and rye. Nucleotide substitution rates in different parts of the Acc-2 gene were assessed. This analysis of the Acc-2 loci provides detailed information regarding evolutionary events at a low--copy-number locus containing important functional genes. These events are likely to be common and to play a significant role in shaping grass genomes.     

A human dihydrofolate reductase intronless pseudogene with an Alu repetitive sequence: multiple DNA insertions at a single chromosomal site

A dihydrofolate reductase (DHFR) pseudogene, hDHFR-psi 3 has been isolated from a human genomic DNA fragment library. Sequence analysis of this gene revealed a lack of introns and the presence of a tract of nine adenines, 90 bp downstream from the end of the coding sequence. These features suggest that hDHFR-psi 3 was derived from a processed RNA molecule that has been converted into DNA and inserted into a chromosome, analogous to the origin of three intronless human DHFR genes previously described. An interesting feature of hDHFR-psi 3 is the presence of a member of the Alu moderately repetitive DNA sequence family within the DHFR coding region. This Alu element is flanked by a 16 bp directly repeated DNA segment derived from DHFR coding sequences. The Alu element apparently has been inserted into the intronless DHFR pseudogene and thus, there have been two insertions at a single chromosomal locus. The hDHFR-psi 3 contains only the 3' half of the DHFR coding sequence. Immediately upstream from the directly repeated sequence before the Alu element is an adenine-rich tract. The DNA farther upstream is moderately repetitive and is related to neither DHFR nor Alu DNA sequence. Therefore, it seems possible that a third insertion has occurred at the same site further disrupting the hDHFR coding sequences.     

A polymerase chain reaction mediated by a single primer: cloning of genomic sequences adjacent to a serotonin receptor protein coding region.

Under appropriate conditions, specific double-stranded DNA product was generated after amplification of genomic DNA sequences in a polymerase chain-like reaction that contained only a single primer. This type of amplification reaction was performed with a variety of primers and substrate DNAs. In addition to nonspecific heterogeneous products, 5 of 11 primers reproducibly directed synthesis of double-stranded DNA that corresponded to the region of the template that contained the authentic primer annealing site. Three of these amplified products were cloned and their ends were sequenced. All three contained a copy of the primer at both 5' ends, and the position of one of the primers represented the authentic primer binding site. In each case, the location of the second copy of the primer indicated that it had initially hybridized to a partially homologous sequence in the template DNA. This single primer reaction makes it possible to amplify and clone a DNA region of unknown sequence that is adjacent to a known DNA sequence. One of the single primer reaction products described here included sequence to the 5' side of the coding region of a serotonin receptor gene that contained a functional promoter.