Detection and resolution of closely related satellite DNA sequences by molecular cloning.

Highly repeated satellite DNAs often consist of mixtures of DNAs with closely related repeating sequences. By cloning individual molecules we have resolved the 1.705 g/cm³ satellite DNA of Drosophila melanogaster into two distinct components: poly$\text{d}\text{A-A-G-A-G}\text{T-T-C-T-C}$ and poly$\text{d}\text{A-A-G-A-G-A-G}\text{T-T-C-T-C-T-C}$. The presence of two distinct sequences within this physically homogeneous satellite DNA had not previously been detected by standard physical, chemical, or sequencing techniques. Both cloning and direct sequence analysis suggest that the five-base-pair and seven-base-pair repeating units reside on separate molecules and are not interspersed with each other.     

Molecular cloning and characterization of endogenous feline leukemia virus sequences from a cat genomic library.

Recombinant bacteriophage lambda clones from a cat genomic library derived from placental DNA of a specific pathogen-free cat were screened to identify endogenous feline leukemia virus (FeLV) sequences. Restriction endonuclease mapping of four different clones indicates that there are a number of similarities among them, notably the presence of a 6.0- to 6.4-kilobase pair (kbp) EcoRI hybridizing fragment containing portions of sequences homologous to the gag, pol, env, and long terminal repeat-like elements of the infectious FeLV. The endogenous FeLV sequences isolated are approximately 4 kbp in length and are significantly shorter than the cloned infectious FeLV isolates, which are 8.5 to 8.7 kbp in length. The endogenous elements have 3.3- to 3.6-kbp deletions in the gag-pol region and approximately 0.7- to 1.0-kbp deletions in the env region. These deletions would render them incapable of encoding an infectious virus and may therefore be related to the non-inducibility of FeLV from uninfected cat cells and the subgenomic expression of these endogenous sequences in placental tissue. It appears that there is conservation in the ordering of restriction sites previously reported in the proviruses of the infectious FeLVs in sequences corresponding to the pol and env boundary as well as the region spanning the env gene of the endogenous clones, whereas a greater divergence occurs among restriction sites mapped to the gag and part of the pol regions of the infectious FeLV. Such deleted, FeLV-related subsets of DNA sequences could have originated either by germ-line integration of a complete ecotropic virus followed by deletion, or by integration of a preexisting, defective, deleted variant of the infectious virus.     

Detection and cloning of expressed sequences linked to a target gene

DNA markers tightly linked to a target gene are essential starting points for positional cloning. We combined ”differential display of mRNA” and ”bulked segregant analysis” in order to detect and clone ten expressed sequences as markers linked to a virus resistance gene in Phaseolus vulgaris. The combination of these two procedures could be used in lieu of positional cloning, provided polymorphisms detectable by differential display exist in the target gene. Isolation of expressed sequences from specific chromosome regions can also be accomplished by combining these procedures. Received: 8 July 1999 / Accepted: 21 March 2000     

Detection and cloning of murine leukemia virus-related sequences from African green monkey liver DNA.

By using low-stringency nucleic acid hybridization conditions and specific subgenomic segments of the AKR ecotropic provirus as probes, murine leukemia virus (MuLV)-related sequences were detected in African green monkey (AGM) liver DNA. The MuLV-reactive segments present in restricted AGM DNA ranged from 1.9 kilobases (kb) to greater than 10 kb in size. On the basis of this finding, a 17-kb segment was cloned from a partial EcoRI AGM library in lambda Charon 4A which shared nearly 5 kb of homology with AKR ecotropic MuLV DNA. The MuLV-related sequences detected in restricted preparations of AGM DNA or present in the cloned monkey DNA reacted with probes mapping 2.0 to 7.0 kb from the 5' terminus of the AKR ecotropic provirus. The AGM clone also contained repeated sequences that flanked the MuLV-related segment. Labeled, subgenomic, MuLV-reactive segments of the monkey clone hybridized to multiple restriction fragments of AGM liver DNA, indicating the presence of several copies of the MuLV-related sequences.     

Retrovirus-related sequences in human DNA: detection and cloning of sequences which hybridize with the long terminal repeat of baboon endogenous virus

Human DNA sequences which hybridized with the long terminal repeats (LTR) of baboon type C virus M7 were detected by non-stringent blot hybridization. About 7 to 10 discrete bands of the LTR-related sequences were commonly observed in the DNAs from four independent human cell lines after digestion with either Eco RI, Hind III or Bam HI. The amounts of these sequences were more abundant in tumor cell lines than in a non-malignant cell line. The human sequences related to the M7 LTR seemed to be located at relatively specific sites on the cell DNA. The human DNA clones which hybridized with M7 LTR were detected in the human DNA library described by Lawn et al. (Cell 15, 1157-1174, 1978), at a frequency of about 300 per haploid genome. Five clones were isolated which shared different extent of homology with M7 LTR and whose restriction maps were totally different one another. The DNA structures of two of them resembled the genome of retroviruses. These results suggest the presence of various types of the LTR-related sequences in human DNA: some of them might represent endogenous virus genomes of human cells.     

A new approach to cloning of tandem repetitive DNA sequences

A new approach to screening of the repeated human DNA sequences tandemly arranged in the genome is described. Efficiency of the developed approach for search of tandemly arranged DNA sequences is corroborated by the obtained experimental data.     

Construction of a new yeast cloning vector containing autonomous replication sequences from Candida utilis.

DNA sequences from the Candida utilis genome which, when cloned into a yeast integration plasmid (YIp5), confer on YIp5 the ability to replicate autonomously in Saccharomyces cerevisiae are described. Several recombinant plasmids which transform S. cerevisiae YNN27 to Ura3+ with an efficiency of 2 X 10(3) transformants per microgram of DNA were obtained. One of the recombinant plasmids, pHMR22 (6.6 kilobases) contains ars (autonomous replication sequence), which is homologous with two different DNA fragments of the C. utilis genome but has no detectable homology to total DNA from Candida albicans, Pachysolen tannophilus, or S. cerevisiae. Restriction and subcloning analyses of pHMR22 showed that Sau3A destroys the functions of cloned ars whereas there are no BamHI, PstI, SalI, HindIII, EcoRI, or PvuII sites in the region of ars which is required for its functional integrity. Thus, pHMR22 appears to be a useful vector for cloning desired genes in S. cerevisiae.     

Amplification and chromosomal dispersion of human endogenous retroviral sequences.

Endogenous retroviral sequences in humans have undergone amplification events involving both viral and flanking cellular sequences. We cloned members of an amplified family of full-length endogenous retroviral sequences. Genomic blotting, employing a flanking cellular DNA probe derived from a member of this family, revealed a similar array of reactive bands in both humans and chimpanzees, indicating that an amplification event involving retroviral and associated cellular DNA sequences occurred before the evolutionary separation of these two primates. Southern analyses of restricted somatic cell hybrid DNA preparations suggested that endogenous retroviral segments are widely dispersed in the human genome and that amplification and dispersion events may be linked.     

Molecular cloning and analysis of the fragile X region in man.

The fragile X syndrome (FraX), the most common inherited form of mental retardation, has been located to Xq27.3. As a step in the molecular analysis of this mutation, we have cloned a contiguous 1.8 Mb region containing the entire fragile X region in YAC and cosmid clones. The cloned area defines a region of 50 kb containing a CpG island, found to be selectively methylated in patients expressing the fragile X phenotype. In this 50kb area we have localised the breakpoints of four somatic cell hybrids selected to break at the position of the fragile site. Fluorescence in-situ hybridisation of cosmids flanking this area shows that the breakpoints, the CpG island and the fragile site coincide.     

Detection of 5-methylcytosine in DNA sequences.

Col E1 DNA has methylated cytosine in the sequence 5'-CC*(A/T)GG-3' and methylated adenine in the sequence 5'-GA*TC-3' at the positions indicated by asterisks(*). When the Maxam-Gilbert DNA sequencing method is applied to this DNA, the methylated cytosine (5-methylcytosine) is found to be less reactive to hydrazine than are cytosine and thymine, so that a band corresponding to that base does not appear in the pyrimidine cleavage patterns. The existence of the methylated cytosine can be confirmed by analyzing the complementary strand or unmethylated DNA. In contrast, the methylated adenine (probably N6-methyladenine) cannot be distinguished from adenine with standard conditions for cleavage at adenine.     

Molecular cloning and sequences of lignin peroxidase genes of Phanerochaete chrysosporium.

The genomic clones encoding lignin peroxidase isozyme H8 and two closely related genes were isolated from Phanerochaete chrysosporium BKM-1767, and their nucleotide sequences were determined. The positions and approximate lengths of introns were found to be highly conserved in all three clones. Analysis of homokaryotic derivatives indicated that the three clones are not alleles of the same gene(s).     

Chimeric plasmids for cloning of deoxyribonucleic acid sequences in Saccharomyces cerevisiae.

Two sets of plasmids, each carrying a Saccharomyces cerevisiae gene and a portion or all of the yeast 2-micron circle linked to the Escherichia coli plasmid pBR322, have been constructed. One of these sets contains a BamHI fragment of S. cerevisiae deoxyribonucleic acid that includes the yeast his3 gene, whereas the other set contains a BamHI fragment of S. cerevisiae that includes the yeast leu2 gene. All plasmids transform S. cerevisiae and E. coli with a high frequency, possess unique restriction endonuclease sites, and are retrievable from both host organisms. Plasmids carrying the 2.4-megadalton EcoRI fragment of the 2-micron circle transform yeast with 2- to 10-fold greater frequency than those carrying the 1.5-megadalton EcoRI fragment of the 2-micron circle. Restriction endonuclease analysis of plasmics retrieved from S. cerevisiae transformed with plasmics carrying the 2.4-megadalton EcoRI fragment showed that in 13 of 96 cases the original plasmic has acquired an additional copy of the 2-mcron circle. These altered plasmids appear to have arisen by means of an interplasmid recombination event while in S. cerevisiae. A clone bank of S. cerevisiae genes based upon one of these composite plasmids has been constructed. By using this bank and selecting directly in S. cerevisiae, the ura3, tyr1, and met2 genes have been cloned.     

Cosuppression of nonhomologous transgenes in Drosophila involves mutually related endogenous sequences.

Cosuppression refers to the phenomenon in which silencing among dispersed homologous genes occurs. Here we demonstrate that two nonhomologous reciprocal fusion genes, white-Alcohol dehydrogenase (w-Adh) and Adh-w, exhibit cosuppression using the endogenous Adh sequence as an intermediary. Deletion of the endogenous Adh gene eliminates the interaction, while reintroduction of an 8.6 kb Adh fragment restores the silencing. Using truncated Adh constructs, a nontranscribed segment in the Adh regulatory region was found to be one of the sequences required for homology recognition. The silencing interaction is initiated during early development. The silenced transgenes are associated with the Polycomb group complex of chromatin proteins.     

Differences between the endogenous and exogenous DNA sequences of Rous-associated virus-O.

DNA sequences related to the endogenous retrovirus of chickens, Rous-associated virus-O (RAV-O), have been examined using site-specific DNA endonuclease analysis of cellular DNA derived from line 15 and line 100 chickens. Individual embryos from both inbred lines were used as a source of embryonic fibroblasts from which cellular DNA was isolated. Analysis of DNA containing either endogenous RAV-O sequences alone or both endogenous and exogenous RAV-O sequences produced identical patterns of RAV-O-specific DNA fragments after digestion with the endonucleases Eco RI, Hind III, BgI II, Bam HI or Xho I. Similar analysis with endonucleases Hinc II or Hha I, however, produced several RAV-O-specific DNA fragments which were derived from cellular DNA containing both endogenous and exogenous RAV-O sequences but not from cellular DNA containing only endogenous sequences. Although some differences exist between the DNA fragments specific for the endogenous viral sequences of line 15 and line 100 cellular DNA, the DNA fragments specific for the exogenous viral sequences were identical between the two inbred lines. Cleavage of an unintegrated linear RAV-O DNA molecule with Hinc II or Hha I produced DNA fragments identical to those specific for the exogenously acquired RAV-O provirus. This suggests that these characteristic fragments contain no cellular DNA. The potential DNA junction fragments containing both viral and cellular DNA, identified after analysis of DNA that contains both endogenous and exogenous viral sequences, were identical to those observed after analysis of DNA containing only endogenous viral sequences. These results support the following conclusions. First, exogenous proviral sequences are integrated into chicken cell DNA following an interaction between viral and cellular DNA that is specific with respect to the virus and nonspecific with respect to the cell. Second, both the free linear RAV-O DNA intermediate and the newly integrated exogenous provirus contain specific endonuclease sites that are not found in endogenous RAV-O DNA sequences. These results suggest that the formation of the exogenous DNA provirus involves specific alteration of the endogenous viral DNA sequences before reinsertion of the sequences as the exogenous RAV-O DNA provirus. It is possible that newly integrated exogenous RAV-O sequences are characterized by specific differences in the pattern of base methylation and a limited sequence arrangement.     

Activation of endogenous retroviral sequences in human leukemia

Endogenous retroviral sequences have been identified in the genomes of several species including humans. Proteins similar to those of primate endogenous viruses have been found on the surface of various malignant cells in man. To further define this role, we have used a primate retrovirus DNA from the Baboon Endogenous Virus to probe a human genomic library for related sequences. A total of 45 clones homologous to BaEV gag-pol were isolated under low stringency hybridization. Of these, several were found to contain DNA which was expressed as RNA at higher levels in human lymphoid leukemic cells than in normal lymphocytes.