Nucleotide sequence of the BamHI repetitive sequence,including the hindIII fundamental unit,as a possible mobile element from the Japanese monkeyMacaca fuscata

Summary Clustered repeat units produced by BamHI digestion of genomic DNA from the Japanese monkeyMacaca fuscata [JMr(BamHI)] were sequenced by dideoxy DNA sequencing. The nucleotide sequences of several individual repeats showed that the BamHI repeat contains the 170-bp HindIII element as an integral part, and that it has more than 90% homology with the HindIII repeat element [AGMr(HindIII)] found in the genomic DNA of the African green monkey. In the JMr(BamHI) repeat unit, the 170-bp HindIII elecment is flanked by a 6-bp inverted repeat, which is part of a 22-bp direct repeat. This latter repeat of 22-bp asymmetrically overlaps the border between the internal AGMr(HindIII)-like region and adjacent regions of the JMr(BamHI) repeat. A similar structural feature of the BamHI repeat unit has been found in the genomic DNA of the baboon, but not in that of the African green monkey. These results show clearly that the BamHI repeat of the modern Japanese monkey originated as a result of insertion of an AGMr(HindIII) element into a certain site(s) of the genomic DNA of an ancestor of the modern Japanese monkey beforeMacaca-Cercocebus divergence.     

The effects of substituted pyrimidines in DNAs on cleavage by sequence-specific endonucleases.

The rates of cleavage of DNAs containing substituents at position 5 of thymine or cytosine have been measured for a variety of sequence-specific endonucleases, so as to determine which features in the DNA sequence are being probed. Phage phi e DNA fully substituted with 5-hydroxymethyluracil is cleaved more slowly by enzymes whose recognition sequences contain A-T base pairs than are DNAs containing thymine, but both types of DNA are cleaved at similar rates by enzymes recognizing sequences composed only of G-C base pairs. Phage PBS2 DNA with uracil completely substituted for thymine is cleaved slowly by several enzymes which recognize sequences containing A-T base pairs (endonucleases Hpa I, HindII, and HindIII), while the rates of cleavage by other enzymes (endonucleases EcoRI and BamHI) are not affected. Phage lambda- and P22 DNAs containing 5-bromouracil are cleaved more slowly by several enzymes (endonucleases HindIII, Hpa I, BamHI) than are thymine-containing DNAs. Enzymes that recognize sequence isomers with the composition G:C:2A:2T (endonucleases EcoRI, Hpa I, HindIII) are not equally affected by substitution at position 5 of thymine, suggesting that they differ in their contacts with A-T base pairs. DNA containing glucosylated 5-hydroxymethylcytosine in place of cytosine is resistant to cleavage by all the endonucleases examined.     

Isolation of a yeast gene, SRH1, that encodes a homologue of the 54K subunit of mammalian signal recognition particle

A 1.7 kilobase HindIII fragment of Saccharomyces cerevisiae DNA was cloned by cross-hybridization with the Escherichia coli secY gene. The complete nucleotide sequence of the 2.6 kb fragment of the yeast genomic DNA containing the cross-hybridizing HindIII fragment was determined. The sequence showed no apparent similarity with that of the E. coli secY gene with the exception of a completely matched sequence of 21 bp, but it contained a 1,623 nucleotide open reading frame coding for a protein of 541 amino acids with a calculated Mr of 59,600. The N-terminal portion of 303 residues of the predicted sequence was homologous to the cytosolic domain of the alpha-subunit of the signal recognition particle receptor (SR alpha), including consensus sequence elements for a GTP binding site, whereas the C-terminal portion of 238 residues had an unusual methionine-rich domain containing several repetitive sequences. An mRNA of 2.0 kb was detected on Northern blotting analysis. The predicted sequence was 48% identical with the reported sequences of the 54K subunit of the mammalian signal recognition particle (SRP54) (Romisch K. et al. (1989) Nature 340, 478-483; Bernstein, H.D. et al. (1989) Nature 340, 482-486). We designated this gene as SRH1 (SRP54 homologue). Gene disruption experiments showed that the SRH1 gene product is essential for cell growth.     

Rearrangement of the surface antigen gene of hepatitis B virus integrated in the human hepatoma cell lines.

The rearrangement of integrated HBV DNA sequences in three different hepatoma cell lines, huH-1, huH-2, KG-55-T from Japanese patients, were studied by blot hybridization using whole HBV genome or a HBsAg or HBcAg DNA as a probe. The characteristic existence of multiple integration sites of HBV DNA sequences in each HindIII-restricted hepatoma cell DNA was revealed by the HBV genome probe. Detection of the isolated HBsAg gene in the HindIII fragment indicates that the integration of HBV DNA was not always related to the maintenance of the whole viral genome, and that movement of the HBsAg gene to another location occurred by rearrangement. On the other hand, the presence of the HBV DNA sequence without the intact HBcAg gene was shown in some of the HindIII fragments, when the HBcAg gene, probe was used, but a HindIII fragment, containing only the HBcAg gene, was not detected so far. The absence of the intact HBcAg gene suggests that the viral genome may lose a part of the HBcAg gene in the process of integration. This is consistent with recent findings of Ogston et al. (1982) that in Woodchuck hepatocellular carcinoma viral sequences are extensively rearranged.     

Long double-stranded sequences (dsRNA-B) of nuclear pre-mRNA consist of a few highly abundant classes of sequences: evidence from DNA cloning experiments.

DNA preparations from about hundred randomly selected clones containing mouse DNA fragments were screened for the existence of sequences complementary to long double-stranded regions of pre-mRNA able to snap back after melting (dsRNA-B). Many clones containing such sequences were found. The cloned sequences can be subdivided into three groups: (1) those complementary to about a half (at least to 30-40%) of the total dsRNA, designated as sequences B1; (2) those complementary to a part of sequence B1; and (3) sequences complementary to about a quarter (at least to 15%) of the total dsRNA referred to as sequence B2. The size of DNA sequence complementary to dsRNA is about 400 base pairs.Melting experiments with hybrids show that the members of B1 family are very similar if not identical, while the divergence among B2 sequences is higher, but still the number of substitutions does not exceed 9% of bases. Thus, the major part of dsRNA-B consists of a small number of highly abundant sequences as was suggested earlier on the basis of renaturation kinetics /1-3/. Sequences B1 and B2 are represented by many copies in the mouse genome and in pre-mRNA, and many of them probably do not form hairpin-like structures.     

Escherichia coli glycerol kinase. Cloning and sequencing of the glpK gene and the primary structure of the enzyme

The glpK gene, which codes for Escherichia coli K-12 glycerol kinase (EC 2.1.7.30, ATP:glycerol 3-phosphotransferase), has been cloned into the HindIII site of pBR322. The gene was contained in a 2.8-kilobase DNA fragment which was obtained from a lambda transducing bacteriophage, lambda dglpK100 (Conrad, C.A., Stearns, G.W., III, Prater, W.E., Rheiner, J.A., and Johnson, J.R. (1984) Mol. Gen. Genet. 195, 376-378). The DNA sequence of 2 kilobases of the cloned HindIII fragment was obtained using the dideoxynucleotide method. The start of the open reading frame for the glpK gene was identified from the N-terminal sequence of the first 22 amino acid residues of the purified enzyme, which was determined by automated Edman degradation. The open reading frame codes for a protein of 502 amino acids and a molecular weight of 56,106 which is in good agreement with the value previously determined by sedimentation equilibrium. The primary structure of the protein as deduced from the gene sequence was corroborated by the isolation and sequencing of four tryptic peptides, which were found to occur at the following amino acid locations: 173-177, 203-211, 279-281, 464-468. The N-terminal sequence of the purified enzyme shows that the enzyme undergoes post-translational processing. Restriction digestion as well as DNA sequencing of the supercoiled plasmid shows that the HindIII fragment is inserted into pBR322 such that the glpK gene is transcribed in a counterclockwise direction. Examination of the upstream DNA sequence reveals two possible promoters of essentially the same efficiency: the P1 promoter of pBR322 and a hybrid promoter which contains both bacterial and pBR322 DNA sequences.     

Saturation of human chromosome 3 with unique sequence hybridization probes

We have generated chromosome 3-specific recombinant libraries in both lambda and cosmid cloning vectors starting with somatic cell hybrids (hamster/human) containing either an intact chromosome 3 or a chromosome 3 with an interstitial deletion removing 75% of long-arm sequences. The libraries contained between 2 X 10(5) and 5 X 10(6) independent recombinants. Approximately 2% of the recombinants in these libraries contain inserts of human DNA. These were identified by hybridizing the recombinants to radioactively labeled total human DNA. Over 2500 recombinants containing human DNA were isolated from these various libraries and DNA was prepared from each of them. This represents 80,000 kb of cloned chromosome 3 sequences. One-third of the DNAs were digested with EcoRI or HindIII, and fragments free of repetitive sequences were radioactively labeled using random hexanucleotide primers and tested as unique sequence hybridization probes. Over 6500 of the fragments were tested and of these 758 were unique sequence probes with minimal or no background hybridization. Their hybridization only to chromosome 3 was verified. These probes, which were derived from 452 independent recombinants, should provide an effective saturation of human chromosome 3.     

Stably bent and low-melting DNA sequences at the origin of replication of chicken alpha-globulin gene domains]

The distribution of specific DNA sequence elements in a 2.9 kb HindIII fragment of chicken DNA containing the replication origin and the upstream matrix attachment site of the alpha-globin gene domain has been studied. The fragment was shown to contain the CR1-type repetitive element and two stable bent DNA sequences.     

Identification of restriction-fragment-length polymorphisms in genomic DNA of the lesser snow goose (Anser caerulescens caerulescens)

A genomic library of partially EcoRI-digested DNA from the lesser snow goose, Anser caerulescens caerulescens, was constructed in the phage vector Charon 4. Phage containing only unique sequences were identified by screening plaques with 32P-labeled genomic DNA. Restriction-fragment- length polymorphisms (RFLPs) were identified by probing DNA from 11-13 male birds from the breeding colony at La Perouse Bay. Of the 17 probes examined, all detected RFLPs with at least one of EcoRi, HindIII, Msp1, and Taq1. Several of them identified highly variable regions with multiple alleles. These RFLPs are valuable DNA markers that can be used for (1) the examination of DNA variation, relatedness, and genetic distance and (2) assessing paternity and maternity. These data suggest that there are higher levels of variation of DNA sequence in birds than had previously been thought to exist.      

Cleavage of Epstein-Barr virus DNA by restriction endonucleases EcoRI, HindIII and BamI.

The cleavage of the DNAs of the B95-8 and P3HR-1 virus strains of Epstein-Barr virus by the restriction endonucleases EcoRI, HindIII and BamI was investigated using a new technique for quantitative evaluation of the fluorescence of ethidium stained DNA fragments separated on agarose gels. The results obtained with B95-8 DNA showed that in addition to the limited repetitions of nucleotide sequences observed in the EcoRI and HindIII cleavage patterns, the molecule contained a BamI fragment with a molecular mass of 2.0 megadaltons which was present in a total of about 11 copies and localized to a limited part of the DNA molecule. The same sequences were also present in the P3HR-1 DNA albeit in a lower molar ratio. P3HR-1 DNA yielded restriction enzyme cleavage patterns suggesting DNA sequence heterogeneity of P3HR-1 virus. No fragment was present in more than about 4 copies per molecule of P3HR-1 DNA. Comparison of the restriction enzyme cleavage patterns of P3HR-1 and B95-8 DNA revealed a high degree of structural homology emphasized by nucleic acid hybridization experiments with EBV complementary RNA synthesized in vitro.     

A large-subunit mitochondrial ribosomal DNA sequence translocated to the nuclear genome of two stone crabs (Menippe)

Two DNA sequences that appear to be homologous to large-subunit mitochondrial ribosomal RNA genes have been identified in the stone crabs Menippe mercenaria and M. adina. Amplification from whole genomic DNA by polymerase chain reaction (PCR) with oligonucleotide primers based on conserved portions of large-subunit mitochondrial rRNA genes consistently amplified two products of similar length (565 and 567 bp). These products differed at 3% of their nucleotide bases, and could be distinguished by a HindIII site. Only one of these sequences (designated the A sequence) was detected by PCR in purified mitochondrial DNA. The other (designated the B sequence) hybridized to total genomic DNA at a level consistent with a nuclear genome location. It is unlikely that the type B product would have been recognized as a nuclear copy by examination of its sequence alone. This is the first report of a mitochondrial gene sequence translocated into the nuclear genome of a crustacean.      

Characterizing the walnut genome through analyses of BAC end sequences

Persian walnut (Juglans regia L.) is an economically important tree for its nut crop and timber. To gain insight into the structure and evolution of the walnut genome, we constructed two bacterial artificial chromosome (BAC) libraries, containing a total of 129,024 clones, from in vitro-grown shoots of J. regia cv. Chandler using the HindIII and MboI cloning sites. A total of 48,218 high-quality BAC end sequences (BESs) were generated, with an accumulated sequence length of 31.2?Mb, representing approximately 5.1% of the walnut genome. Analysis of repeat DNA content in BESs revealed that approximately 15.42% of the genome consists of known repetitive DNA, while walnut-unique repetitive DNA identified in this study constitutes 13.5% of the genome. Among the walnut-unique repetitive DNA, Julia SINE and JrTRIM elements represent the first identified walnut short interspersed element (SINE) and terminal-repeat retrotransposon in miniature (TRIM) element, respectively; both types of elements are abundant in the genome. As in other species, these SINEs and TRIM elements could be exploited for developing repeat DNA-based molecular markers in walnut. Simple sequence repeats (SSR) from BESs were analyzed and found to be more abundant in BESs than in expressed sequence tags. The density of SSR in the walnut genome analyzed was also slightly higher than that in poplar and papaya. Sequence analysis of BESs indicated that approximately 11.5% of the walnut genome represents a coding sequence. This study is an initial characterization of the walnut genome and provides the largest genomic resource currently available; as such, it will be a valuable tool in studies aimed at genetically improving walnut.     

Cytogenetic and molecular characterization of a highly repeated DNA sequence in the peach potato aphid Myzus persicae

Electrophoresis following digestion of Myzus persicae genomic DNA with HindIII showed the presence of a prominent band of approximately 200 bp whereas a faint electrophoretic band corresponding to DNA fragments of about 3000 bp was observed after digestion with ApaI. In situ digestion with restriction enzymes, followed by in situ nick translation, showed that ApaI targets are localized at the nucleolus organizer-bearing X telomeric region, whereas HindIII restriction sites are clustered in intercalary C-positive areas on the same X chromosome. Fluorescent in situ hybridization (FISH) carried out by using digoxygenin-labeled HindIII repeats as probe fully confirmed overlapping between the hybridization sites of this probe and the AT-rich intercalary heterochromatic bands on the X chromosome. These findings, together with published data, allow us to conclude that the M. persicae genome possesses three classes of C-positive heterochromatin: (i) a GC-rich argentophilic band located on one telomere of the X chromosome that contains ApaI targets; (ii) AT-rich intercalary bands located on the X chromosome containing clustered HindIII fragments; (iii) AT-rich telomeric bands, located on autosomes, consisting of HaeIII repeats. Molecular analysis has shown that the length of the HindIII repeat consensus sequence is 189 bp with an AT content of 67%. Southern blotting with HindIII monomers revealed a regular ladder of bands composed of multimers of basic length that are characteristic of satellite DNAs. The HindIII repeat displays other features typical of eukaryotic satellite arrays such as overlapping with heterochromatic bands and a high degree of sequence similarity among monomers (84%–94%). A similarity plot showed that sequences were particularly variable in the 50–100 bp region whereas they proved to be highly conservative in the first 50 bp, thus suggesting that this portion of the repeat might be functionally important. Received: 23 February 1999; in revised form: 21 July 1999 / Accepted: 28 July 1999     

The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase in Euglena gracilis chloroplast DNA: location, polarity, cloning, and evidence for an intervening sequence

The gene for the large subunit (LS) of ribulose-1,5,-bisphosphate carboxylase of Euglena gracilis Z chloroplast DNA has been mapped by heterologous hybridization with DNA restriction fragments containing internal sequences from the Zea mays and Chlamydomonas reinhardii LS genes. The Euglena LS gene which has the same polarity as the Euglena rRNA genes has been located with respect to Pst I, Pvu I, and HindIII sites within the Eco RI fragment Eco A. The region of Euglena chloroplast DNA complementary to an 887 bp internal fragment from the Chlamydomonas chloroplast LS gene is interrupted by a 0.5-1.1 kbp non-complementary sequence. This is the first chloroplast protein gene located on the Euglena genome, and the first evidence for an intervening sequence within any chloroplast protein gene.     

Transcriptional antitermination activity of the synthetic nut elements of coliphage lambda. I. Assembly of the nutR recognition site from boxA and nut core elements

An active nutR antiterminator was reconstructed from two synthetic modules, one containing the 8-bp boxA (5'-CGCTCTTA) and the other the 17-bp nutR core (5'-AGCCCTGAAAAAGGGCA) sequence. The modules were synthesized with HindIII cohesive ends, which upon annealing and ligation created an 8-bp spacer (5'-CAAAGCTT) between the boxA and nutR core. The 8-bp length was the same as in the native nutR (5'-CACATTCC), but the sequence showed less than 38% homology. The antitermination mediated by the synthetic nutR, was 68-80% efficient when tested in the pp-nutR-N-tL1-galK expression plasmid, analogous to that used by Drahos and Szybalski [Gene, 16 (1981) 261-274]. The cloned boxA by itself has no activity, while the nutR core alone shows only marginal (5-10%) antiterminator function. Increasing the distance between boxA and the nutR core from 8 bp to 20-28 bp, i.e., by one to two turns of the DNA helix (about 10 bp per turn), has little effect on the antiterminator function, whereas use of spacers with length about halfway between 8 and 20 bp results in reduced antitermination. It appears that both the sequences and spacial arrangement of the boxA and nut elements are important for efficient antiterminator function.     

Detailed analysis of the portion of the herpes simplex virus type 1 genome encoding glycoprotein C.

We previously showed that the right third of HindIII fragment L (0.59 to 0.65) of herpes simplex virus type 1 (HSV-1) encodes a family of mRNAs some members of which appear to be related by splicing. In the experiments described in this communication, we determined the nucleotide sequence of the DNA encoding this mRNA family and precisely located the mRNAs associated with this DNA sequence. The major mRNA species is unspliced and encoded by a 2.520-nucleotide region. Just upstream of the 5' end are TATA and CAT box sequences characteristic of HSV-1 promoters. The 3' end maps near a region containing a nominal polyadenylation signal. Three minor species (2,400, 2,200, and 1,900 bases, respectively) appear to share a very short leader sequence with the 5' end of the major mRNA and are then encoded by uninterrupted DNA sequences beginning about 100, 400, and 625 bases downstream of the 5' end of the major unspliced mRNA. These positions map at or very near positions which agree reasonably well with consensus splice acceptor sequences. The fourth mRNA is encoded by a contiguous 730-nucleotide sequence at the 3' end of the major unspliced mRNA and has its 5' end just downstream of recognizable TATA and CAT box sequences. We suggest that this mRNA is controlled by its own promoter. The nucleotide sequence data, in combination with the mRNA localization, demonstrate four potential polypeptides encoded by the region. The largest is 1,569 bases long and defines a 523-amino acid protein with sequence features characteristic of a glycoprotein. This was confirmed to be HSV-1 glycoprotein C by immune precipitation of the in vitro translation product of the major unspliced mRNA, performed with a polyspecific antibody to HSV-1 envelope glycoproteins (anti-env-1 serum), and by comparison of tryptic peptides of this translation product with those of authentic HSV-1 glycoprotein C. Polypeptides encoded by some of the minor species also were tentatively identified.