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Biomedical subjects

H Delius

Publications and source records attributed to H Delius.

At least 91 records · Page 5Linked to original sources
PubMed1985

Separation of complementary strands of plasmid DNA using the biotin-avidin system and its application to heteroduplex formation and RNA/DNA hybridizations in electron microscopy.

A method for the separation of complementary strands with the help of the biotin-avidin system is described. Restriction fragments were terminally labeled at both ends with biotinylated nucleotides. The DNA was cut by a second restriction enzyme, and the fragments were bound to an avidin agarose column. The non-biotinylated strands were eluted with 0.1 M NaOH, and the biotin-labeled strands were subsequently released from the column by elution with 50% guanidine isothiocyanate/formamide. Contamination of the separated strands by complementary single strands was less than 4%.-Separated linear single strands of the vector pEMBL were prepared. On annealing with recombinant circular DNA a substitution loop is formed which provides position and orientation markers for the unambiguous electron microscopic analysis of heteroduplexes or hybrids formed with the inserted sequences. -The terminal biotin label was visualized by complex formation with a streptavidin-ferritin conjugate.

Avidin↗
PubMed1985

Isolation and characterization of the chicken cardiac myosin light chain (L-2A) gene. Evidence for two additional N-terminal amino acids.

The contractile proteins of striated muscle are encoded by multigene families and constitute an excellent system to investigate differentiation and developmental control of gene expression. Different forms of myosin light chains are expressed in skeletal muscle as well as in the myocard. To study the gene structure and molecular mechanisms underlying differential gene expression, the structural cardiac myosin light chain 2 (MLC-2A) gene was isolated from a chicken genomic DNA library. Restriction enzyme mapping, electron microscopic analysis, and partial sequencing revealed that the gene coding for the MLC mRNA of 700 nucleotides in length extends over 4.2 kilobases of DNA and is interrupted by 5 introns. Sequence analysis led to the detection of two codons for additional amino acids at the N terminus which were not reported to be present in the mature protein and are presumably removed post-translationally. These two amino acids, methionine and alanine, are coded on two separate exons split by the largest intron of the entire gene. Southern blot analysis of genomic chicken DNA indicates the presence of one MLC-2A gene per haploid chicken genome.

Amino Acid Sequence↗
PubMed1985

The structure of precursor mRNAs and of excised intron RNAs in chloroplasts of Euglena gracilis.

Partially spliced precursor mRNAs (pre-mRNAs) in the steady-state population of RNA from chloroplasts of Euglena gracilis were found by electron microscopy. The structure and the frequency of the pre-mRNAs of the psbA gene (the gene for the 32-kd protein of photosystem II), which is split by four introns in Euglena chloroplasts was analysed by electron microscopy. A chloroplast DNA (cpDNA) fragment containing the psbA gene from Euglena, was cloned into a pEMBL vector. The single-stranded recombinant phage DNA of the coding strand was prepared and hybridized with cpRNA. The majority of hybrids were formed with mature mRNA, but approximately 8% of the hybrids were formed with pre-mRNAs. The pre-mRNAs were either unspliced or incompletely spliced. A detailed analysis of the structure and the frequency of the pre-mRNAs of the psbA gene showed that the four introns are neither spliced out in a strictly random way, nor in a 5'-3' or 3'-5' direction. Introns 2 and 3 are preferentially spliced out first, intron 1 intermediately and intron 4 is generally spliced out last. However, this sequence is not a strict rule. We conclude that the introns can be spliced independently, each one at a different rate. The coding strand from a fragment of the psbA gene was separated and annealed with low mol. wt. cpRNA, which was isolated from an agarose gel. Small circular hybrids were found at the positions of the four introns, demonstrating for the first time covalently closed circular excised intron RNAs (iRNAs) in chloroplasts.

Animals↗
PubMed1985

A putative transforming gene of Jijoye virus differs from that of Epstein-Barr virus prototypes.

The P3HR-1 strain of Epstein-Barr virus (EBV), a nontransforming clonal derivative of Jijoye (EBV), is characterized by a deletion of 6.6 kb involving part of the BamHI-W repeats and the adjacent region including the NotI repeats. In the transforming parental Jijoye virus this region differs from the corresponding regions in B95-8 or M-ABA virus. The HindIII-B fragments which carry this region from both Jijoye and prototype M-ABA (EBV) viruses have been cloned and subclones have been constructed which contain the left-hand part of HindIII-B from the HindIII to the BglII site (BglII-delta C fragment). By restriction enzyme analysis the inserts were found to be of equal size (6.3 kb) but to differ in their restriction enzyme pattern. Heteroduplexes formed under stringent conditions in the presence of T4 gene 32 protein revealed a substitution loop of 1750 +/- 200 nucleotides. Heteroduplex formation under nonstringent conditions showed that the substituted sequences are partially homologous to each other, with the regions of nonhomology confined to three distinct areas of 100 to 200 nucleotides. The partial homology observed between both regions indicates that they have evolved from a common ancestor. By hybridization of a Jijoye virus subclone containing only sequences of the substituted region to Northern blots a 2.8-kb polyadenylated transcript was detected indicating that the substituted region is expressed in Jijoye cells.

Cell Line↗
PubMed1985

Analysis of cosmids using linearization by phage lambda terminase.

A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage lambda terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage lambda (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.

Animals↗
PubMed1985

Molecular cloning and physical mapping of the tupaia herpesvirus genome.

Purified virion DNA of about 200 kilobase pairs of tupaia herpesvirus strain 2 was cleaved with EcoRI or HindIII restriction endonuclease. Restriction fragments representing the complete viral genome including both termini were inserted into the EcoRI, HindIII, and EcoRI-HindIII sites of the bacterial plasmid pAT153. Restriction maps for the restriction endonucleases EcoRI and HindIII were constructed with data derived from Southern blot hybridizations of individual viral DNA fragments or cloned DNA fragments which were hybridized to either viral genome fragments or recombinant plasmids. The analysis revealed that the tupaia herpesvirus genome consists of a long unique sequence of 200 kilobase pairs and that inverted repeat DNA sequences of greater than 40 base pairs do not occur, in agreement with previous electron microscopic data. No DNA sequence homology was detectable between the tupaia herpesvirus DNA and the genome of murine cytomegalovirus, which was reported to have a similar structure. In addition, seven individual isolates of tupaia herpesvirus were characterized. The isolates can be grouped into five strains by their DNA cleavage patterns.

Animals↗
PubMed1985

Characterization of the DNA of canine adenovirus by restriction enzyme analysis.

The DNA of canine adenovirus (canAV 1, strain Behring H.c.c. 269) was characterized in detail. The molecular weight of canAV DNA was (20.1 +/- 0.1) x 10(6) daltons as determined by contour length measurements, and 19.9 x 10(6) daltons as determined by restriction enzyme analysis. A terminal protein was found to be covalently linked to the 5' ends of canAV DNA. Physical maps of viral DNA were constructed for BamHI, ClaI, SacI, SalI, and SmaI enzymes. Three international strains of canAV [American Type Culture Collection (ATCC) catalogue No. VR-800, VR-293, and VR-133] were analyzed under the same conditions. CanAV 1 (ATCC VR-133 and VR-293) had DNA cleavage patterns identical to the H.c.c. 269 strain, whereas the DNA cleavage pattern of canAV 2 (ATCC VR-800) was clearly different.

Adenoviridae↗
PubMed1984

Transcription strategy of coronaviruses: fusion of non-contiguous sequences during mRNA synthesis.

MHV replicates in the cell cytoplasm and viral genetic information is expressed in infected cells as one genomic sized RNA ( mRNA1 ) and six subgenomic mRNAs. The seven RNAs were assumed to have common 3' ends of the size of RNA7 , the smallest RNA. The data reported here, show that this model is too simple and that the mRNAs are composed of a leader and body sequence. Electron microscopic analysis of hybrids formed between single stranded cDNA copied from mRNA7 and genomic RNA or mRNA6 shows that genomic RNA, mRNA6 and mRNA7 have common 5' terminal sequences. Furthermore, nucleotide sequence analysis shows that the nucleotide sequence of the 5' end of mRNA7 diverges from the corresponding region of the genome just upstream from the initiation codon of the nucleocapsid gene. Because the synthesis of each mRNA is inactivated by UV irradiation in proportion to its own length, the subgenomic mRNAs are apparently not produced by the processing of larger RNAs. The available data have to be explained by translocation of the polymerase/leader complex to specific internal positions on the negative strand. In this way the leader and body sequences are joined together by a mechanism completely different from conventional RNA splicing but nevertheless giving the same end result.

Animals↗
PubMed1984

Two deletions in the Epstein-Barr virus genome of the Burkitt lymphoma nonproducer line Raji.

The Epstein-Barr virus genome carried in the Burkitt lymphoma nonproducer cell line Raji was characterized by partial denaturation mapping and by hybridization of cloned viral fragments to filters containing separated Raji DNA fragments. Partial denaturation mapping revealed that the EBV DNA population of Raji cells is homogeneous and that two deletions are observed in distant parts of the genome compared to linear DNA isolated from virus particles of different strains. These deletions were characterized by blot analysis. One deletion of 3.15 kb lies within HindIII-E; the second is 2.4 kb and is located close to the right terminus of linear viral DNA. The two deletions were observed in several cell lines derived from the Raji line. These deletions might contribute to the inability of Raji cells to produce EBV either spontaneously or upon induction.

Burkitt Lymphoma↗
PubMed1984

Nine introns with conserved boundary sequences in the Euglena gracilis chloroplast ribulose-1,5-bisphosphate carboxylase gene.

The single, chloroplast encoded gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) from Euglena gracilis is found to contain nine intervening sequences. The intervening sequences were identified by heteroduplex analysis between Euglena rbcL and the non-intron-containing rbcL from Spinacea oleracea, by electron microscopy of Euglena rbcL DNA-mRNA hybrids, and by cloning, restriction endonuclease analysis, and partial DNA sequencing. The identification, locus, and coding properties for six of ten exons was confirmed by partial DNA sequence analysis. Each of the nine introns in the approximately 6.5 kb rbcL locus is approximately 0.5 kb in length. The DNA sequence of five 3'-intron/5'-exon and four 3'-exon/5'-intron boundaries are highly conserved. A proposed consensus sequence is (formula; see text) These conserved sequences could play a role in an mRNA splicing mechanism in chloroplasts analogous to that in eucaryotic nuclei.

Base Sequence↗
PubMed1984

Intervening sequences in chloroplast genomes.

Chloroplast DNAs from the green algae Euglena gracilis and from broad beans (Vicia faba) were hybridized with homologous chloroplast RNA. The hybrids were analyzed by electron microscopy. Almost all transcribed regions of the Euglena genome, except for the rRNA genes, contain intervening sequences. A minimum of 50 introns was observed amounting to about 32 kb of the 145 kb genome. Only four spliced transcripts with a total number of six introns (about 0.8 kb each) were detected in the bean chloroplast RNA. A complete transcription map of the Euglena chloroplast genome was established. Transcribed regions occur on the strand that codes for the rRNAs (plus strand) as well as on the complementary strand (minus strand). The polarity of transcription reverses at least six times.

Base Sequence↗
PubMed1984

Genetic recombination can generate altered restriction specificity.

A recombinant strain, isolated following the transduction of an Escherichia coli recipient carrying the Salmonella typhimurium (SB) specificity genes with DNA from a donor having the Salmonella potsdam (SP) specificity, was shown [Bullas, L.R., Colson, C. & Van Pel, A. (1976) J. Gen. Microbiol. 95, 166-172] to have neither SB nor SP specificity but to encode a novel restriction specificity, SQ. The heteroduplex analysis of the hsdS (specificity) genes of the SB and SP restriction and modification systems described here identifies a conserved sequence of around 100 base pairs flanked by two nonhomologous regions each of approximately 500 base pairs. This organization parallels that previously deduced from the DNA sequences of the hsdS genes of the related E. coli K-12, B, and D restriction systems. The present heteroduplex analyses further show that the hsdS gene conferring the SQ specificity derives one nonhomologous region from the SB gene and the other from the SP gene, as predicted from genetic exchange within the conserved sequence. This finding supports the idea that two domains of an hsdS polypeptide, which are different for each specificity, may correlate with two regions of the DNA sequence recognized. It has been shown that the recognition sequences for E. coli K-12 and B each consist of two short oligonucleotide sequences interrupted by a nonspecific sequence. A similar organization is suggested for the Salmonella specificity systems, providing the potential for evolutionary diversification of restriction specificities as a result of recombination within the conserved sequence of the hsdS gene.

Bacteriophage lambda↗
PubMed1984

Insertion of retrovirus into the first intron of alpha 1(I) collagen gene to embryonic lethal mutation in mice.

Integration of the Moloney murine leukemia virus (M-MuLV) into the germ line of Mov-13 mice blocked formation of stable alpha 1(I) collagen mRNA and led to an embryonic lethal mutation. A 14-kilobase fragment representing the integration site of the virus was molecularly cloned and identified as the alpha 1(I) collagen gene. Sequence and nuclease S1 mapping analyses were performed to characterize the position of the proviral genome in relation to the transcriptional map of the mutated gene. The results indicated that the virus has inserted into the first intron 19 base pairs downstream of the intron/exon boundary. Sequence comparison showed a striking homology of exon sequences and sequences up to 215 base pairs upstream of the mRNA start between the mouse and the human alpha 1(I) collagen gene. This indicates that the sequences upstream of the mRNA start are highly conserved during evolution, suggesting that this region has an important role in the control of tissue-specific collagen expression.

Amino Acid Sequence↗
PubMed1984

DNA analysis of insect iridescent virus 6: evidence for circular permutation and terminal redundancy.

DNA analysis of small insect iridovirus 6 was performed. Combined exonuclease-restriction endonuclease digestions revealed that all resulting fragments were degraded without preference for any one DNA fragment. Upon denaturation and reannealing of native linear Chilo iridescent virus DNA (158 x 10(6) daltons), duplex DNA circles of a smaller size (140 x 10(6) daltons) with protruding tails were formed.

Journal Article↗
PubMed1983

Analysis of the genome of fish lymphocystis disease virus isolated directly from epidermal tumours of pleuronectes.

Virions of fish lymphocystis disease virus (FLDV), a member of the iridovirus family, were isolated directly from lymphocystis disease lesions of individual flatfishes and purified by sucrose and subsequent cesium chloride gradient centrifugation to homogeneity as judged by electron microscopy. The isolated FLDV DNAs appear to be heterogeneous in size. Contour length measurements of 43 DNA molecules gave an average length of 49 +/- 23 microns, corresponding to 93 +/- 44 X 10(6) D. Molecular weight estimations of FLDV DNA by restriction enzyme analysis resulted in only 64.8 X 10(6) D indicating an excess length of the DNA of about 50%. FLDV DNA was sensitive to lambda 5'-exonuclease and to E. coli 3'-exonuclease III without preference of any one terminal DNA restriction fragment. Denaturation and reannealing experiments of FLDV DNA resulted in the formation of circular DNA molecules of 34.25 microns contour length (= 65.22 X 10(6) D). This result suggests that FLDV DNA contains directly repeated sequences at both ends and that it is terminally redundant. FLDV DNA is methylated in cytosine. FLDV DNA did not hybridize with frog virus DNA indicating that the two iridoviruses are not closely related to each other. Restriction enzyme analysis and Southern blot hybridizations revealed that FLDV isolates can be classified into two different strains: FLDV strain 1 occurs in flounders and plaice, whereas strain 2 is usually found in lesions of dabs.

Animals↗
PubMed1983

Structure of the hepatitis B virus genome.

The extent and position of the single-stranded gap in DNA molecules from Dane particles isolated from two donors of the adw serotype were determined by molecular hybridization and electron microscopic methods. The results showed that in each preparation more than 99% of the circular molecules are of uniform length and contain both single- and double-stranded regions. They confirmed that one end of the short strand is fixed with respect to the single EcoRI site within the molecule and to the nick in the long strand, but they also showed that although the position of the other end is variable, there is a preferred minimum length of about 650 to 700 nucleotides for the single-stranded region.

DNA, Viral↗
PubMed1983

Coronavirus mRNA synthesis involves fusion of non-contiguous sequences.

Positive-stranded genomic RNA of coronavirus MHV and its six subgenomic mRNAs are synthesized in the cytoplasm of the host cell. The mRNAs are composed of leader and body sequences which are non-contiguous on the genome and are fused together in the cytoplasm by a mechanism which appears to involve an unusual and specific 'polymerase jumping' event.

Base Sequence↗