Physical mapping of the early region of bacteriophage T7 DNA.
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Centrifugation, Density Gradient↗
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Plastid origin of an extrachromosomal DNA molecule from Plasmodium, the causative agent of malaria.
Several species of Plasmodium have been shown to contain a circular extrachromosomal DNA molecule which is widely supposed to be mitochondrial DNA. However, it has recently been shown to have a number of features in common with chloroplast DNA. Here, a phylogenetic analysis of RNA polymerase coding sequences from the Plasmodium molecule has been carried out using distance matrix, maximum likelihood, parsimony and operator invariant methods. The analysis indicates that the molecule is in fact derived from an oxygenic photosynthetic organism and should be regarded as plastid DNA. This suggests that Plasmodium originated from a phototroph that has lost the capacity to photosynthesize.
Amino Acid Sequence↗
Structural studies of the lysozyme coded by the pneumococcal phage Cp-1. Conformational changes induced by choline.
The CPL-1 lysozyme coded by the pneumococcal phage Cp-1 has been overproduced in Escherichia coli under the control of a modified lipoprotein lactose promoter. This result has provided the conditions to analyse the CPL-1 secondary structure by circular dichroism (CD). The CD spectra recorded in the far-ultraviolet region showed, at neutral pH, two minima at 210 nm and 230 nm and a shoulder at 217 nm, whereas two bands at 260 nm and 295 nm were observed in the near-ultraviolet region. It has been estimated, by using the CDPROT program, that the protein is composed of 19% alpha-helix, 32% beta-sheet, 28% beta-turn and 21% random coil. Minor changes in the CD spectra were detected either when the pH was varied over 6-10 or when the ionic strength was increased to 1 M NaCl. Choline, a well known modulator of the enzyme activity that is present in the pneumococcal cell wall, induced remarkable changes in the intensities of the bands at 210, 230 and 295 nm, with the appearance of an unusual positive band at 225 nm. The conformational change was reversible and correlated with the competitive inhibitory effect of choline on the lysozyme activity, supporting, by a new and direct experimental approach, the basic role of choline in the recognition of the cell wall substrate. The analyses of the secondary structure prediction and the CD data reported here are compatible with the two-domain structure of CPL-1 reinforce our hypothesis that the C-terminal region is directly involved in the binding of the enzyme to the pneumococcal teichoic and lipoteichoic acids.
Amino Acid Sequence↗
Physical and genetic characterization of the genome of Lactobacillus lactis bacteriophage LL-H.
Bacteriophage LL-H is a virulent phage of Lactobacillus lactis LL23. A restriction map of the phage genome was constructed with various restriction endonucleases. This chromosome has a 34-kilobase size and seems to be circularly permuted. We used a bank of LL-H restriction fragments to study the expression of five of the seven main phage particle proteins. Immunoblotting experiments permitted the mapping on the chromosome of several genes coding for phage particle proteins. We also show that the gene of the main capsid protein is expressed from its own promoter in an Escherichia coli strain.
Bacteriolysis↗
Viroids: molecular infectious agents.
In 1971, unique small RNA molecules, the viroids, were found to cause specific infectious diseases of plants. They are the smallest and simplest contagious agents known. Until now, 14 viroids have been described and 12 diseases of potatoes, tomatoes, citruses, chrysanthemums, cucumbers, hops, coconut palms avocado trees and burdock are known to be caused by viroids. The common symptoms of these diseases are: stunting of plants. discoloration of veins, epinasty, curling and distortions of leaves, chlorotic or necrotic spots etc., followed by death of the diseased plants. All viroids are ssRNAs of m.w. ranging from 1.1 x 10(5) to 1.7 x 10(5), corresponding to chains of just 246 to 371 ribonucleotides. For 10 viroids, complete nucleotide sequences are known PSTV, CSV, CEV, TPMV and TASV show 60%-80% homology with each other; in analogy, ASBV, HSV, CPFV. GV and CCCV are closely homologous to each other, too, but just distantly related to the PSTV group. Extensive intramolecular base pairing creates a characteristic secondary structure of the cyclic viroid RNA chain, native viroids appearing as quasi double-stranded, unbranched, very short rod-like structures with short single-stranded loops. (Thus PSTV forms rods about 50 nm long and 2 nm wide.) The stretch of nearly all viroids bears a common central conserved region of 19 bp. The "upper" part of this region is, presumably, the cleavage-ligation site of viroid oligomers during replication. Viroids are located and replicated in nuclei of infected cells, in association with their nucleoli. Their replication is directed by host DNA-dependent RNA polymerase II using cRNA oligomers as templates according to the rolling circle model. Viroid RNA has no mRNA function. The virulence of viroids is coded by their virulence modulating region in the "left hand" part of their molecules: a single nucleotide substitution between nucleotides 43 and 56 within this region alters the virulence. Most probably, viroids have originated by the circularization of spliced-out transcripts of eucaryotic introns. A stable complex may be created between the 5' end of U1 snRNA and nucleotides 257 to 279 of PSTV cRNA strand; thus the pathogenic effects of viroids seem to be a result of their interference with pre-mRNA processing.
Base Sequence↗
Cloning and sequencing of the genome of spiroplasma virus 4.
Spiroplasma virus 4 (Sp V4) has a circular single-stranded DNA. The replicative form (RF) of Sp V4 has been purified from infected cells of Spiroplasma melliferum, strain G1, and cloned in Escherichia coli (HB101) using plasmid pBR328 as the vector. The cloned RF was shown to be infectious by transfection. The Sp V4 RF was randomly subcloned in E. coli (TG1) using the M1 3 mp8 RF as the vector and sequenced by the dideoxy chain termination technique. We found that UGA is probably not a termination codon, but codes for tryptophan. Eight open reading frames, including that for the 65,000-dalton capsid protein, have been detected; they involve all three reading frames.
Bacteriophages↗
Plastid DNA from Pyrenomonas salina (Cryptophyceae): physical map, genes, and evolutionary implications.
Cryptomonads are thought to have arisen from a symbiotic association between a eukaryotic flagellated host and a eukaryotic algal symbiont, presumably related to red algae. As organellar DNAs have proven to be useful tools in elucidating phylogenetic relationships, the plastid (pt) DNA of the cryptomonad alga Pyrenomonas salina has been characterized in some detail. A restriction map of the circular 127 kb ptDNA from Pyrenomonas salina was established. An inverted repeat (IR) region of about 5 kb separates two single-copy regions of 15 and 102 kb, respectively. It contains the genes for the small and large subunit of rRNA. Ten protein genes, coding for the large subunit of ribulose-1,5-bisphosphate carboxylase, the 47 kDa, 43 kDa and 32 kDa proteins of photosystem II, the ribosomal proteins L2, S7 and S11, the elongation factor Tu, as well as the alpha- and beta-subunits of ATP synthase, have been localized on the restriction map either by hybridization of heterologous gene probes or by sequence homologies. The gene for the plastidal small subunit (SSUr) RNA has been sequenced and compared to homologous SSU regions from the cyanobacterium Anacystis nidulans and plastids from rhodophytes, chromophytes, euglenoids, chlorophytes, and land plants. A phylogenetic tree constructed with the neighborliness method and indicating a relationship of cryptomonad plastids with those of red algae is presented.
Base Sequence↗
Complete nucleotide sequence of the Bacillus thuringiensis subsp. israelensis plasmid pTX14-3 and its correlation with biological properties.
The complete nucleotide sequence of the plasmid pTX14-3 from Bacillus thuringiensis subsp. israelensis has been determined. The circular DNA molecule was 7649 bp and had a G + C content of 35.1%. Twenty-two open reading frames larger than 50 codons were identified. Ten of these open reading frames are suggested to be protein coding regions. The existence of the polypeptides encoded by the mob14-3 and rep14-3 genes were verified by maxi-cells analysis in Escherichia coli. Even though the rep14-3 gene was expressed in E. coli the plasmid pTX14-3 was unable to replicate in this bacterium. The minimal region of the plasmid pTX14-3 required for replication in B. thuringiensis was identified. Potential secondary structures upstream of the rep14-3 gene indicated regulation by antisense RNA and transcription attenuation. Extensive sequence homology with the B. thuringiensis subsp. thuringiensis plasmid pGI2 was found in the last part of the mob14-3 gene, downstream of the rep14-3 gene, and in the region containing the single-strand origin of replication (i.e., the minus origin) of pTX14-3. A sequence of 700 bp containing multiple direct repeats was found in an ORF encoding a glycine and proline rich protein of 35.9 kDa. 1.2 kbp upstream and 0.1 kbp downstream of this ORF was found a large direct repeat of 230 bp (87% identity). The region between this direct repeat was often spontaneously deleted from plasmid derivatives containing the entire pTX14-3.
Amino Acid Sequence↗
Novel transcripts from the Ultrabithorax domain of the bithorax complex.
We present a detailed analysis of the transcriptional products of the bithoraxoid (bxd) region of the Ultrabithorax domain in the bithorax complex of Drosophila melanogaster. This region is transcribed twice during development: between 3 and 6 hr of embryogenesis, a set of early transcripts, 1.1 to 1.3 kb in size, is synthesized; from the midthird larval instar through the adult stages, a late 0.8-kb transcript is synthesized. We have sequenced five cloned cDNAs representing early transcripts and three cDNAs representing the late transcript and have located their exons within the 40 kb of DNA comprising the bxd region. S1 nuclease protection and primer extension of both the early and late transcripts were used to further elucidate their structure. The early RNAs are produced by complex differential splicing of a series of exons derived from a 26-kb primary transcript. Curiously, these RNAs do not possess significant protein coding potential. The late bxd RNA comprises a single exon transcribed from an intronic region of the early transcription unit. This RNA, by contrast, possesses excellent coding potential and, if translated, would yield a 101-amino-acid polypeptide.
Animals↗
Bacteriophage lambda; abortive infection of bacteria lysogenic for phage P2.
The efficiency of plating of wild-type lambda on a host lysogenic for P2 is less than 10(-6), and only a small number of infected cells produce progeny phage. Lambda can adsorb and inject its DNA normally in such cells; the DNA can circularize and is not nicked or degraded, but replication is severely impaired. Mutants of P2, which as prophages no longer interfere with lambda, have been isolated and found to be recessive to wild type, implying that P2 prophage codes for a diffusible product involved in lambda interference. The P2 gene product responsible for preventing lambda growth also kills recombination-deficient bacteria of the recB and recC classes under conditions where P2 does not normally kill the host. Mutants of lambda that are resistant to interference are recessive to wild-type lambda. Thus lambda actively participates in its own interference. The lambda-mutants that are resistant to interference are unable to synthesize at least two nonessential proteins. In addition, they are unable to grow on recombination-deficient bacteria of the recA class, but they can grow on recA recB double mutants.
Carbon Isotopes↗
Structure and expression of a gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL) in the colourless euglenoid flagellate Astasia longa.
A gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) was identified on a circular 73 kb DNA from the colourless euglenoid flagellate Astasia longa. The rbcL gene of Astasia extends over 3968 bp. It is a split gene interrupted by seven introns as compared to nine intervening sequences in the rbcL gene of the phylogenetically related Euglena gracilis. Coding sequences as well as the positions of the introns within this gene are highly conserved in comparison with the Euglena rbcL except that two introns are missing in Astasia. The alignment of the amino acid sequences deduced from the nucleotide sequences of rbcL of Astasia and Euglena shows 82% identical amino acids whereas 15% of the amino acids represent conservative changes. A 1.5 kb transcript of the rbcL gene was revealed by northern blot analysis of Astasia RNA. By immunoblot analysis the gene product of rbcL was detected as a 53 kDa polypeptide. Genes for components of the chloroplast transcriptional and translational systems encoded by chloroplast DNA of plants and green algae are conserved on the 73 kb DNA of Astasia [24, 25, 26]. From our finding that Astasia obviously is capable of synthesizing the Rubisco large subunit one must conclude that these genes are expressed and form functional plastid transcriptional and translational systems.
Amino Acid Sequence↗
Characterization of cloned chicken anemia virus DNA that contains all elements for the infectious replication cycle.
Circular double-stranded replication intermediates were identified in low-molecular-weight DNA of cells of the avian leukemia virus-induced lymphoblastoid cell line 1104-X-5 infected with chicken anemia virus (CAV). To characterize the genome of CAV, we cloned linearized CAV DNA into the vector pIC20H. Transfection of the circularized cloned insert into chicken cell lines caused a cytopathogenic effect, which was arrested when a chicken serum with neutralizing antibodies directed against CAV was added. Chickens inoculated at 1 day of age with CAV collected from cell lines transfected with cloned CAV DNA developed clinical signs of CAV. The 2,319-bp cloned CAV DNA contained all the genetic information needed for the complete replication cycle of CAV. The CAV DNA sequence has three partially overlapping major reading frames coding for putative peptides of 51.6, 24.0, and 13.6 kDa. The CAV genome probably contains only one promoter region and only one poly(A) addition signal. Southern blot analysis using oligomers derived from the CAV DNA sequence showed that infected cells contained double- and single-stranded CAV DNAs, whereas purified virus contained only the minus strand. It is the first time that the genome of one of the three known single-stranded circular DNA viruses has been completely analyzed.
Amino Acid Sequence↗
A salt bridge stabilizes the helix formed by isolated C-peptide of RNase A.
C-peptide, which contains the 13 NH2-terminal residues of RNase A, shows partial helix formation in water at low temperature (1 degree C, pH 5, 0.1 M NaCl), as judged by CD spectra; the helix is formed intramolecularly [Brown, J. E. & Klee, W. A. (1971) Biochemistry 10, 470-476]. We find that helix stability depends strongly on pH: both a protonated histidine (residue 12) and a deprotonated glutamate (residue 9 or 2 or both) are required for optimal stability. This information, together with model building, suggests that the salt bridge Glu-9- ... His-12+ stabilizes the helix. Formation of the helix is enthalpy driven [van't Hoff delta H, - 16Kcal/mol (1 cal = 4.18 J)] and the helix is not observed above 30 degrees C. Proton NMR data indicate that several side chains adopt specific conformations as the helix is formed. These results have two implications for the mechanism of protein folding. First, they indicate that short alpha-helices, stabilized by specific side-chain interactions within the helix, can be stable enough in water to function as folding intermediates. Second, they suggest that similar experiments with peptides of controlled amino acid sequence could be used to catalogue the intrahelix interactions that stabilize or destabilize alpha-helices in aqueous solution. These data might provide the code relating amino acid sequence to the locations of alpha-helices in proteins.
Chromatography, High Pressure Liquid↗
The nucleotide sequence of the infectious cloned DNA components of potato yellow mosaic virus.
The complete nucleotide sequence of a Venezuelan isolate of potato yellow mosaic virus (PYMV) has been determined, showing it to be typical of subgroup I geminiviruses in that it is whitefly-transmitted, has a circular, bipartite ssDNA genome and possesses bidirectionally orientated open reading frames (ORFs). The two genomic components have little sequence similarity apart from a common region of 268 nucleotides (nt) which is almost identical. Analysis of ORFs revealed six potential coding regions encoding proteins of Mr greater than 10K, four in PYMV A (2593 nt) and two in PYMV B (2547 nt), which are preceded by regulatory transcription elements and have polyadenylation signals present at the ends. Amino acid sequence alignments of PYMV DNA ORF-encoded proteins with those encoded by other previously sequenced geminivirus ORFs show that PYMV is closely related to those geminiviruses isolated from the New World, especially in the putative coat protein gene regions.
Amino Acid Sequence↗
Targeted disruption of the Myxococcus xanthus orotidine 5'-monophosphate decarboxylase gene: effects on growth and fruiting-body development.
The Myxococcus xanthus gene coding for orotidine 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23) was cloned. The M. xanthus uraA gene efficiently complemented an Escherichia coli OMP decarboxylase mutant, permitting it to grow in the absence of uracil. Electroporation of M. xanthus with a circular plasmid carrying a selectable uraA::kan gene disruption resulted in homologous recombination at the chromosomal uraA locus. Chromosomal integration of the gene disruption plasmid created heterozygous (uraA+/uraA::kan) tandem duplications. These tandem duplications were unstable and segregated auxotrophic uraA::kan daughters at frequencies of 2 x 10(-4) to 8 x 10(-4) per viable cell. Rare uraA::kan segregants were easily obtained by selecting for resistance to the toxic analog 5-fluoroorotic acid. Our experiments suggest that the cloned uraA gene could facilitate the use of gene duplications in the genetic analysis of M. xanthus development. The uraA mutants could utilize uracil, uridine, or uridine 5'-phosphate for growth, indicating that M. xanthus has pyrimidine salvage pathways. During multicellular development, uraA::kan gene disruption mutants sporulated to wild-type levels but formed smaller and more numerous aggregates than did their uraA+ parent, regardless of whether uracil was added to the medium. Pyrimidine deprivation of uraA mutants, under conditions that otherwise supported vegetative growth, failed to induce fruiting-body development or sporulation.
Cloning, Molecular↗
Theoretical and experimental approach to recognition of amino acid by tRNA and nucleotide II.
Strong and specific interactions between amino acids and their cognate tRNAs have been found by the CD, fluorescence, and UV absorption difference spectroscopy and by the gel-filtration method. New types of strong hydrogen bonding interactions between an amino acid and polynucleotides, nucleotides, nucleosides, and nucleic acid bases in aqueous solution have also been detected. These findings are strong experimental evidence for the C4N model of the genetic code. The cases of glutamate and aspartate will theoretically be discussed in details.
Amino Acids↗
Introns, protein syntheses and aging.
In the fungus Podospora, a correlation has recently been established between the presence of circular DNA molecules arising from the mitochondrial genome (SEN-DNAs) and the senescence syndrome. Here, I propose a hypothesis which accounts for the initial event which leads to the first SEN-DNA. A molecule in the most frequent situation where the SEN-DNA is an intron which might code for a maturase. This hypothesis is based upon several observations made either in Podospora or in the yeast S. cerevisiae. It assumes that mitochondrially synthesized maturases are unspecific nucleases able to work at the level of RNA and DNA molecules. Their specificity for RNA splicing instead of DNA is given by cytoplasmic proteins. Therefore, if the balance between cytoplasmic and mitochondrial protein syntheses is disturbed in favour of the mitochondrial compartment, the maturase would be accumulated and allowed to splice introns from DNA instead of RNA molecules. This hypothesis can account for aging of higher eucaryotic cells by postulating analogous processes in their nuclear compartment.
Ascomycota↗
Secondary rearrangements and post-rearrangement selection contribute to restricted immunoglobulin DJH expression in young rabbit bone marrow.
Extrachromosomal circular DNA purified from bone marrow cells of 2-weeks-old rabbits was assayed by polymerase chain reaction to determine the relative rearrangement frequencies of immunoglobulin DH to JH genes in vivo. DH genes rearranged to individual JH genes with different frequencies. This bias did not correlate with potential sequence overlaps in the DH or JH coding sequences. The JH2 and JH4 genes were the preferred targets of recombination in primary rearrangements. Although primary rearrangements to JH6 were relatively infrequent, secondary rearrangements were detected. This assay also revealed previously undescribed JH pseudogenes with functional recombination signal sequences. Analyses of genomic VDJH indicated that B cells expressing VDJH4 heavy chains survived and dominated in the bone marrow environment due to secondary rearrangements and/or post-rearrangement selection.
Amino Acid Sequence↗