Molecular Cloning of α-Amylase Genes from DROSOPHILA MELANOGASTER. I. Clone Isolation by Use of a Mouse Probe

A cloned alpha-amylase cDNA sequence from the mouse is homologous to a small set of DNA sequences from Drosophila melanogaster under appropriate conditions of hybridization. A number of recombinant lambda phage that carry homologous Drosophila genomic DNA sequences were isolated using the mouse clone as a hybridization probe. Putative amylase clones hybridized in situ to one or the other of two distinct sites in polytene chromosome 2R and were assigned to one of two classes, A and B. Clone lambda Dm32, representing class A, hybridizes within chromosome section 53CD. Clone lambda Dm65 of class B hybridizes within section 54A1-B1. Clone lambda Dm65 is homologous to a 1450- to 1500-nucleotide RNA species, which is sufficiently long to code for alpha-amylase. No RNA homologous to lambda Dm32 was detected. We suggest that the class B clone, lambda Dm65, contains the functional Amy structural gene(s) and that class A clones contain an amylase pseudogene.     

Isolation and chromosomal localization of genomic DNA sequences coding for cytoplasmic superoxide dismutase from Drosophila melanogaster

A synthetic oligodeoxynucleotide 18-mer probe derived from the amino acid sequence of Drosophila melanogaster cytoplasmic superoxide dismutase (cSOD) was used to screen a D. melanogaster genomic library. One of the positive clones maps by in situ hybridization to position 68A8-9 on the left arm of polytene chromosome 3, the region to which cSOD mutants have previously been mapped genetically. Partial sequence analysis verifies the presence of cSOD-coding sequences in this clone and indicates that the intron structure of the Drosophila cSOD gene differs significantly from its human counterpart.     

Isolation and characterization of cloned DNA sequences containing ribosomal protein genes of Drosophila melanogaster.

Ribosomal (r) proteins encoded by polyadenylated RNA were specifically precipitated in vitro from polysomes by using antibodies raised against characterized Drosophila melanogaster r proteins. The immuno-purified mRNA in the polysome complex was used to prepare cDNA with which to probe a D. melanogaster genomic library. Selected recombinant phages were used to hybrid select mRNAs, which were analyzed by in vitro translation. Three clones containing the genes for r proteins 7/8, S18, and L12 were positively identified by electrophoresis of the translation products in one-dimensional and two-dimensional polyacrylamide gels. Sequences encoding r proteins S18 and L12 were found to be present in the genome in single copies. In contrast, the polynucleotide containing the region encoding 7/8 may be repeated or may contain or be flanked by short repeated sequences. The sizes of mRNAs that hybridized to the recombinant clone containing 7/8 were significantly larger than would be expected from the molecular weight of protein 7/8, implying that there were unusually long 5' and 3' noncoding sequences. The mRNAs for r proteins S18 and L12 were however, only about 10% larger. In situ hybridizations to salivary gland polytene chromosomes, using the recombinant phage, revealed that the recombinant clone containing the gene for r protein 7/8 hybridized to 5D on the X chromosome; the recombinant clone containing the gene for S18 hybridized to 15B on the same chromosome, and the recombinant phage containing the gene for L12 hybridized to 62E on chromosome 3L. It is of interest that the genomic locations of all three r protein clones were within the chromosomal intervals known to contain the Minute mutations [M(1)0, M(1)30, and M(3)LS2]. Although each clone contained sequences specifying two to four proteins, none had more than one identifiable r protein gene, suggesting that different D. melanogaster r protein genes may not be closely linked.     

Characterization and developmental expression of beta tubulin genes in Drosophila melanogaster.

Genomic clones containing beta tubulin sequences were isolated from a lambda library of Drosophila melanogaster. In situ hybridization localized three genes to 56D and 60B on chromosome 2 as well as to 85D on chromosome 3. The latter was known through genetic analysis to be specifically expressed during spermatogenesis. The genomic clone, pTu85, derived from this region contains one complete beta tubulin coding region as well as the 3' end of an additional so far unidentified beta tubulin gene. Genomic Southern hybridizations reveal a total of five fragments with beta tubulin homology. Clone pTu56 codes for an RNA of 1.8 kb which is expressed in all developmental stages. Clone pTu60 codes for a 2.5-kb RNA expressed during embryogenesis and pupation. In testes RNA we detected a 2.2-kb message homologous to pTu85.     

Chromosome localization of the lambda20p1.4 clone of the Drosophila melanogaster nuclear lamina DNA in the melanogaster species subgroup of the genus Drosophila (Sophophora)

Chromosome localization of sequences homologous to lambda 20p1.4 of the Drosophila melanogaster nuclear lamina DNA (nlDNA) was established by in situ hybridization in species of the melanogaster subgroup. DNA of the lambda 20p1.4 clone was shown to be located in the chromocenter in all the species examined. Laboratory strains of D. simulans, D. mauritiana, and D. sechellia exhibited interspecific differences in localization of lambda 20p1.4 nlDNA on chromosome arms. In eight natural populations, intraspecific polymorphism of lambda 20p1.4 nlDNA chromosome localization was shown to be present in D. simulans but absent in D. melanogaster. The possible participation of transposable elements in nlDNA relocation is discussed.     

Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene.

Monoclonal antibodies were prepared against a fraction of nuclear proteins of Drosophila melanogaster identified as tightly binding to DNA. Four of these antibodies were directed against a 19-kilodalton nuclear protein; immunofluorescence staining of the polytene chromosomes localized the antigen to the alpha, beta, and intercalary heterochromatic regions. Screening of a lambda gt11 cDNA expression library with one of the monoclonal antibodies identified a recombinant DNA phage clone that produced a fusion protein immunologically similar to the heterochromatin-associated protein. Polyclonal sera directed against the bacterial lacZ fusion protein recognized the same nuclear protein on Western blots. A full-length cDNA clone was isolated from a lambda gt10 library, and its DNA sequence was obtained. Analysis of the open reading frame revealed an 18,101-dalton protein encoded by this cDNA. Two overlapping genomic DNA clones were isolated from a Charon 4 library of D. melanogaster with the cDNA clone, and a restriction map was obtained. In situ hybridization with these probes indicated that the gene maps to a single chromosome location at 29A on the 2L chromosome. This general strategy should be effective for cloning the genes and identifying the genetic loci of chromosomal proteins which cannot be readily assayed by other means.     

Specific DNA sequences are associated with nuclear envelopes of pseudonurse cells in Drosophila melanogaster otu 11

Nuclei of ovarian pseudonurse cells from the mutant strain of Drosophila melanogaster otu 11 are suitable for mapping the attachment of chromosomes to the nuclear envelope (NE). Loci in contact with the NE included region 20CD of the X chromosome, region 41 of chromosome 2, the proximal end of region 81 of chromosome 3, and region 101 of chromosome 4. In situ hybridization revealed that all 4 regions contained sequences homologous to clone lambda20p1.4. DNA of clone lambda20p1.4 was previously found to bind specifically to purified D. melanogaster lamins. These results suggest that specific DNA sequences are involved in attachment of chromosomes to NE in vivo.     

Direct and general selection for lysogens of Escherichia coli by phage lambda recombinant clones.

We report a simple in vivo technique for introducing an antibiotic resistance marker into phage lambda. This technique could be used for direct selection of lysogens harboring recombinant phages from the Kohara lambda bank (a collection of ordered lambda clones carrying Escherichia coli DNA segments). The two-step method uses homologous recombination and lambda DNA packaging to replace the nonessential lambda DNA lying between the lysis genes and the right cohesive (cos) end with the neomycin phosphotransferase (npt) gene from Tn903. This occurs during lytic growth of the phage on a plasmid-containing host strain. Neomycin-resistant (npt+) recombinant phages are then selected from the lysates containing the progeny phage by transduction of a polA1 lambda lysogenic host strain to neomycin resistance. We have tested this method with two different Kohara lambda phage clones; in both cases, neomycin resistance cotransduced with the auxotrophic marker carried by the lambda clone, indicating complete genetic linkage. Linkage was verified by restriction mapping of purified DNA from a recombinant phage clone. We also demonstrate that insertion of the npt+ recombinant phages into the lambda prophage can be readily distinguished from insertion into bacterial chromosomal sequences.     

The expression and genomic organization of randomly selected cloned Drosophila melanogaster genes

A lambda recombinant DNA library containing Drosophila melanogaster nuclear DNA inserts was screened with cDNA made from oocyte and gastrula poly(A)+ RNA. 124 clones were isolated which represented sequences complementary to a distribution of abundancies of their RNAs. The clone set was then used as probes to identify those whose RNA abundancies changed during embryonic development. The vast majority of clones showed little difference during development. Four different clones were identified whose poly(A)+ RNAs were quantitatively regulated; two were oocyte-specific, and two were embryonic-specific. 44 clones were chosen for in situ hybridization to salivary gland polytene chromosomes. The location and distribution of their sites are described. A class of clones, identified by in situ hybridization to the nucleolus, is further described. These clones contain a scrambled array of ribosomal intervening sequences.     

Organization of gene and non-gene sequences in micronuclear DNA of Oxytricha nova.

In order to study the derivation of the macronuclear genome from the micronuclear genome in Oxytricha nova micronuclear DNA was partially digested with EcoRI, size fractionated, and then cloned in the lambda phage Charon 8. Clones were selected a) at random b) by hybridization with macronuclear DNA or c) by hybridization with clones of macronuclear DNA. One group of these clones contains only unique sequence DNA, and all of these had sequences that were homologous to macronuclear sequences. The number of macronuclear genes with sequences homologous to these micronuclear clones indicates that macronuclear sequences are clustered in the micronuclear genome. Many micronuclear clones contain repetitive DNA sequences and hybridize to numerous EcoRI fragments of total micronuclear DNA, yielding similar but non-identical patterns. Some micronuclear clones containing these repetitive sequences also contained unique sequence DNA that hybridized to a macronuclear sequence. These clones define a major interspersed repetitive sequence family in the micronuclear genome that is eliminated during formation of the macronuclear genome.     

A Ty3/gypsy retrotransposon-like sequence localizes to the centromeric regions of cereal chromosomes

A 745 bp sequence (pSau3A9) located at the centromeres of several cereal species was isolated from a sorghum BAC library by Jiang et al. (1996, Proc. Natl Acad. Sci. USA, 93, 14210-14213). We have amplified a partially homologous 809 bp sequence from barely genomic DNA by PCR and localized it to the centromeres of barley, wheat and rye chromosomes by fluorescent in situ hybridization (FISH). Sequence analysis showed this barley homolog of pSau3A9 to have high similarity to the integrase region of the polyprotein gene of Ty3/gypsy group retrotransposons. Using this integrase sequence as a probe, several clones were isolated from a lambda library constructed of genomic barley DNA. One of the lambda clones contained coding regions for all five catalytic sites characteristic of the retrotransposon polyprotein. Two direct repeats flanking the polyprotein gene are homologous to the cereal centromeric sequence described by Aragón-Alcaide et al. (1996, Chromosoma, 105, 261-268) and may represent all or part of the long-terminal repeats (LTRs). Different plasmid subclones containing various regions of the lambda clone were used in FISH to show that the entire polyprotein gene and upstream flanking sequences, including the presumed LTR, are present at barley centromeres. The preferential (or exclusive) localization of an apparently complete retroelement within the centromeric regions of several cereal species raises interesting questions about its role in karyotype evolution and centromere function.     

Drosophila melanogaster has only one myosin alkali light-chain gene which encodes a protein with considerable amino acid sequence homology to chicken myosin alkali light chains.

A chimeric lambda DNA molecule containing the myosin alkali light-chain gene of Drosophila melanogaster was isolated. The encoded amino acid sequence was determined from the nucleic acid sequence of a cDNA homologous to the genomic clone. The identity of the encoded protein was established by two criteria: (i) sequence homology with the chicken alkali light-chain proteins and (ii) comparison of the two-dimensional gel electrophoretic pattern of the peptides synthesized by in vitro translation of hybrid-selected RNA to that of myosin alkali light-chain peptides extracted from Drosophila myofibrils. There is only one myosin alkali light-chain in D. melanogaster; its chromosomal location is region 98B . This gene is abundantly expressed during the development of larval as well as adult muscles. The Drosophila protein appears to contain one putative divalent cation-binding domain (an EF hand) as compared with the three EF hands present in chicken alkali light chains.     

A complete and a truncated U1 snRNA gene of Drosophila melanogaster are found as inverted repeats at region 82E of the polytene chromosomes.

A phage containing two sequences homologous to U1 snRNA was isolated from a Drosophila melanogaster genomic library, and identified with a previously cloned D. melanogaster U1 snRNA gene. DNA sequence analysis showed that complete and truncated U1 snRNA genes are present, both of which have base substitutions relative to U1 snRNA. These genes show conservation of 5' and 3' flanking regions relative to other U1 and U2 snRNA genes of Drosophila. Intramolecular renaturation experiments and electron microscope mapping demonstrates that the two U1 snRNA sequences are present as inverted repeats about 2.7kb apart, separated by a smaller pair of inverted repeats of an unrelated sequence. These U1 snRNA sequences were located by in situ hybridization at 82E, and related sequences were found at 21D and 95C on the polytene chromosome map. The results are discussed with reference to the origin and function of snRNAs.     

Novel structure of a human U6 snRNA pseudogene

A genomic DNA library containing human placental DNA cloned into phage lambda Charon 4A was screened for snRNA U6 genes. In vitro 32P-labeled U6 snRNA isolated from HeLa cells was used as a hybridization probe. A positive clone containing a 4.6-kb EcoRI fragment of human chromosomal DNA was recloned into the EcoRI site of pBR325 and mapped by restriction endonuclease digestion. Restriction fragments containing U6 RNA sequences were identified by hybridization with isolated U6[32P]RNA. The sequence analysis revealed a novel structure of a U6 RNA pseudogene, bearing two 17-nucleotide(nt)-long direct repeats of genuine U6 RNA sequences arranged in a head-to-tail fashion within the 5' part of the molecule. Hypothetical models as to how this type of snRNA U6 pseudogene might have been generated during evolution of the human genome are presented. When compared to mammalian U6 RNA sequences the pseudogene accounts for a 77% overall sequence homology and contains the authentic 5'- and 3'-ends of the U6 RNA.     

Isolation and structural mapping of a human c-src gene homologous to the transforming gene (v-src) of Rous sarcoma virus.

We have utilized a lambda Charon 4A human genomic library to isolate recombinant clones harboring a highly conserved c-src locus containing nucleotide sequences homologous to the transforming gene of Rous sarcoma virus (v-src). Four overlapping clones spanning 24 kilobases of cellular DNA were analyzed by restriction endonuclease mapping. Human c-src sequences homologous to the entire v-src region are present in a 20-kilobase region that contains 11 exons as determined by restriction mapping studies utilizing hybridization to labeled DNA probes representing various subregions of the v-src gene and by preliminary DNA sequencing analyses. A considerable degree of similarity exists between the organization of the human c-src gene and that of the corresponding chicken c-src gene with respect to exon size and number. However, the human c-src locus is larger than the corresponding chicken c-src locus, because many human c-src introns are larger than those of chicken c-src. alu family repetitive sequences are present within several human c-src introns. This locus represents a highly conserved human c-src locus that is detectable in human cellular DNAs from various sources including placenta, HeLa cells, and WI-38 cells.     

Characterization of the feline c-abl proto-oncogene

Analysis of total feline DNA by genomic blot hybridization, using the viral oncogene of Abelson murine leukemia virus as a specific probe, has led to the identification of multiple v-abl homologous genetic sequences in the cat genome. Upon restriction endonuclease BamHI digestion, the combined size of the v-abl homologous DNA fragments was about 31 kbp. To characterize these sequences further, four independent v-abl homologous cosmid clones with overlapping cellular inserts have been isolated from a gene library of cat lung genomic DNA. These inserts represent a contiguous region of cellular DNA sequences of 56 kbp in length. Within this region of the feline genome, the v-abl homologous sequences are discontinuously dispersed over a region of about 34 kbp. They represent the complete feline v-abl cellular homolog and are colinear with the viral v-abl oncogene. Nine regions of highly repetitive DNA sequences have been mapped in close proximity to v-abl homologous sequences. These results establish the presence of only a single c-abl proto-oncogene in the cat genome and present its genetic organization.     

Isolation and characterization of the complete chicken beta-globin gene region: frequent deletion of the adult beta-globin genes in lambda.

A library of bacteriophage lambda clones containing chicken chromosomal DNA was screened, using the adult beta-globin cDNA plasmid pHb 1001 as a probe. Sixteen overlapping clones were isolated containing 35 kilobase pairs (kbp) of chicken DNA. Characterization of these clones revealed four beta-like globin genes, some genomically repeated sequences, but no pseudo-genes. The four beta-like genes have an average intergenic distance of less than half of that found for the mammalian beta-like globin gene clusters so far characterized. The overall features of the map were confirmed by genomic Southern analysis. Frequent deletions were shown to occur between the various beta-like globin genes during phage propagation. The presumptive hatching gene in particular was always associated with abnormal lambda clones although we were able to find one such clone that did contain a normal copy of the hatching gene itself. Probably such deletions explain the failure to recover this gene in previous attempts.