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PubMed1990

Delta hepatitis. The disease and the virus.

Delta hepatitis virus is a new human pathogen always found associated with hepatitis B virus (HBV) causing both fulminant hepatitis and the accelerated progression of pre-existing HBV hepatitis. The virus is coated in HBsAg and contains circular single stranded RNA genome with very high intramolecular base-pairing, similar to the genomes of plant viroids, and the HDV antigen, a specific marker of HDV infection also found in the nuclei of infected hepatocytes. The genome is about 1700 nucleotides long and of minus polarity with a major constant antigenomic Open Reading Frame (ORF) which codes for the antigen. Replication of viral RNA appears to proceed by a rolling circle mechanism and specific self-cleavage and self-ligation of both genomic and antigenomic HDV RNA strands has been demonstrated in vitro. HDV appears to require HBsAg for its propagation and hepatotropism but not to depend on HBV for replication.

Animals↗
PubMed2026

The complete Chloroplast Genome of Dianthus Helenae, an Endemic Species with Medicinal Potential from the Nuratau Mountains, Uzbekistan.

Dianthus helenae Vved. is an endemic medicinal species of the Nuratau Mountains, Uzbekistan, and its genomic resources have remained largely unavailable. In this study, we sequenced, assembled, and characterized the complete chloroplast genome of D. helenae and evaluated its phylogenetic position within Dianthus. The plastome exhibited a typical circular quadripartite structure with a total length of 149,567 bp, comprising a large single-copy (LSC) region of 82,856 bp, a small single-copy (SSC) region of 17,105 bp, and a pair of inverted repeats (IRs) of 24,803 bp each. The genome contained the typical set of chloroplast genes, including protein-coding genes, transfer RNAs, and ribosomal RNAs, with duplicated genes located in the IR regions. Phylogenetic analysis based on complete chloroplast genome sequences strongly supported the placement of D. helenae within Dianthus and recovered it as a distinct lineage relative to other sampled species. Sliding window analysis of nucleotide diversity revealed uneven sequence variation across the plastome, with higher variability in the SSC and LSC regions than in the IRs. Several highly variable loci, including trnK-UUU , rps16-trnQ-UUG , rpl32, ycf1, and ndh-associated regions, were identified as potential molecular markers. These results provide an important genomic resource for Dianthus and establish a foundation for future phylogenetic, taxonomic, conservation, and molecular identification studies of this endemic Central Asian species.

Genome, Chloroplast↗
PubMed1992

V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes.

Lymphoid cells from scid mice initiate V(D)J recombination normally but have a severely reduced ability to join coding segments. Thymocytes from scid mice contain broken DNA molecules at the TCR delta locus that have coding ends, as well as molecules with signal ends, whereas in normal mice we previously detected only signal ends. Remarkably, these coding (but not signal) ends are sealed into hairpin structures. The formation of hairpins at coding ends may be a universal, early step in V(D)J recombination; this would provide a simple explanation for the origin of P nucleotides in coding joints. These findings may shed light on the mechanism of cleavage and suggest a possible role for the scid factor.

Animals↗
PubMed1992

Structural organization of the chloroplast genome of the chromophytic alga Vaucheria bursata.

The chloroplast genome of the chromophytic alga Vaucheria bursata has been characterized by restriction site and gene mapping analysis. It is represented by a circular molecule 124.6 kb in size. An inverted sequence duplication (IR) not larger than 5.85 kb carries the rRNA genes and separates two single-copy regions of 64.6 kb and 48.3 kb from one another. The Vaucheria plastid genome exists in two equimolar isomers which is due to intramolecular flip-flop recombination within the IR sequences. The coding sites for 21 structural and soluble proteins have been mapped on both single-copy regions using heterologous gene sequences as probes. Although the overall gene order is found to be rearranged when compared with other chromophytic algal and land plant chloroplast genomes, most of the transcriptional units of cyanobacteria and land plant chloroplast genomes appear to be conserved. The phylogenetic implications of these findings are further discussed.

Chloroplasts↗
PubMed1998

DNA as a vehicle for the self-assembly model of computing.

A DNA version of the self-assembly model of computing, feasible using currently available laboratory techniques, is proposed. Input signals are coded into unmethylated and methylated oligonucleotides which then hybridize with a backbone that contains complementary sequences. Different input signal patterns are thus represented as DNA duplexes with distinctly different conformational dynamics, in particular different equilibria of B and Z DNA. The pattern classification activity of the system is mediated by the interactions that lead to the secondary structural organization. Circular dichroism may be used for readout.

Computer Simulation↗
PubMed1998

Mutation detection and single-molecule counting using isothermal rolling-circle amplification.

Rolling-circle amplification (RCA) driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. In the presence of two primers, one hybridizing to the + strand, and the other, to the - strand of DNA, a complex pattern of DNA strand displacement ensues that generates 10(9) or more copies of each circle in 90 minutes, enabling detection of point mutations in human genomic DNA. Using a single primer, RCA generates hundreds of tandemly linked copies of a covalently closed circle in a few minutes. If matrix-associated, the DNA product remains bound at the site of synthesis, where it may be tagged, condensed and imaged as a point light source. Linear oligonucleotide probes bound covalently on a glass surface can generate RCA signals, the colour of which indicates the allele status of the target, depending on the outcome of specific, target-directed ligation events. As RCA permits millions of individual probe molecules to be counted and sorted using colour codes, it is particularly amenable for the analysis of rare somatic mutations. RCA also shows promise for the detection of padlock probes bound to single-copy genes in cytological preparations.

Alleles↗
PubMed1976

The divergence between human and baboon globin genes.

Complementary DNA (cDNA) was prepared with viral RNA-directed DNA polymerase from purified baboon globin messenger RNA (mRNA). Homologous and heterologous hybrids between human and baboon mRNAs and cDNAs were compared for extent of hybridisation and thermal stability. Higher mRNA inputs to the hybridizations were required to reach saturation in the heterologous cases. The melting temperature of the heterologous hybrid was 5 degrees C lower than the homologous hybrid. Between these two primates, divergence has occurred in the globin gene to a smaller extent than that possible from third position changes in the coding sequences of the divergence of total DNA. Globin cDNA prepared from baboon will not in general be useful as a probe for human globin mRNA or human globin gene sequences.

Animals↗
PubMed1978

Construction of a novel plasmid-phage hybrid: use of the hybrid to demonstrate ColE1 DNA replication in vivo in the absence of a ColE1-specified protein.

A hybrid bacteriophage, P420, was constructed in vitro; it contains part of bacteriophage P4 and a 3.6-kilobase derivative of plasmid ColE1. In Escherichia coli, the plasmid-phage hybrid can exist as a stable plasmid or can be packaged into infective bacteriophage particles. Replication of P420, directed by the ColE1 replicon, was found to occur after P420 phage infection of E. coli cells that had been incubated in the presence of chloramphenicol. Replication began without a lag period and resulted in the synthesis of covalently closed circles of P420 DNA. Like ColE1 DNA replication but unlike that of P4, replication was dependent on DNA polymerase I and was sensitive to rifampicin. The presence of a resident ColE1 plasmid in the infected cells resulted in an inhibition of the replication of the incoming P420 DNA. These results indicate that ColE1 does not require a plasmid-coded protein to replicate its DNA in vivo and demonstrate the utility of P4 bacteriophage for coupling bacteriophage properties to a plasmid replicon.

Bacterial Proteins↗
PubMed1985

Concordance of experimentally mapped or predicted Z-DNA sites with positions of selected alternating purine-pyrimidine tracts.

The recent electronmicroscopic and biochemical mapping of Z-DNA sites in phi X174, SV40, pBR322 and PM2 DNAs has been used to determine two sets of criteria for identification of potential Z-DNA sequences in natural DNA genomes. The prediction of potential Z-DNA tracts and corresponding statistical analysis of their occurrence have been made on a sample of 14 DNA genomes. Alternating purine and pyrimidine tracts longer than 5 base pairs in length and their clusters (quasi alternating fragments) in the 14 genomes studied are under-represented compared to the expectation from corresponding random sequences. The fragments [d(G X C)]n and [d(C X G)]n (n greater than or equal to 3) in general do not occur in circular DNA genomes and are under-represented in the linear DNAs of phages lambda and T7, whereas in linear genomes of adenoviruses they are strongly over-represented. With minor exceptions, potential Z-DNA sites are also under-represented compared to random sequences. In the 14 genomes studied, predicted Z-DNA tracts occur in non-coding as well as in protein coding regions. The predicted Z-DNA sites in phi X174, SV40, pBR322 and PM2 correspond well with those mapped experimentally. A complete listing together with a compact graphical representation of alternating purine-pyrimidine fragments and their Z-forming potential are presented.

Animals↗
PubMed1995

Sequential interactions of structural proteins in phage phi 29 procapsid assembly.

The mechanism of viral capsid assembly is an intriguing problem because of its fundamental importance to research on synthetic viral particle vaccines, gene delivery systems, antiviral drugs, chimeric viruses displaying antigens or ligands, and the study of macromolecular interactions. The genes coding for the scaffolding (gp7), capsid (gp8), and portal vertex (gp10) proteins of the procapsid of bacteriophage phi 29 of Bacillus subtilis were expressed in Escherichia coli individually or in combination to study the mechanism of phi 29 procapsid assembly. When expressed alone, gp7 existed as a soluble monomer, gp8 aggregated into inclusion bodies, and gp10 formed the portal vertex. Circular dichroisin spectrum analysis indicated that gp7 is mainly composed of alpha helices. When two of the proteins were coexpressed, gp7 and gp8 assembled into procapsid-like particles with variable sizes and shapes, gp7 and gp10 formed unstable complexes, and gp8 and gp10 did not interact. These results suggested that gp7 served as a bridge for gp8 and gp10. When gp7, gp8, and gp10 were coexpressed, active procapsids were produced. Complementation of extracts containing one or two structural components could not produce active procapsids, indicating that no stable intermediates were formed. A dimeric gp7 concatemer promoted the solubility of gp8 but was inactive in the assembly of procapsid or procapsid-like particles. Mutation at the C terminus of gp7 prevented it from interacting with gp8, indicating that this part of gp7 may be important for interaction with gp8. Coexpression of the portal protein (gp20) of phage T4 with phi 29 gp7 and gp8 revealed the lack of interaction between T4 gp20 and phi 29 gp7 and/or gp8. Perturbing the ratio of the three structural proteins by duplicating one or another gene did not reduce the yield of potentially infectious particles. Changing of the order of gene arrangement in plasmids did not affect the formation of active procapsids significantly. These results indicate that phi 29 procapsid assembly deviated from the single-assembly pathway and that coexistence of all three components with a threshold concentration was required for procapsid assembly. The trimolecular interaction was so rapid that no true intermediates could be isolated. This finding is in accord with the result of capsid assembly obtained by the equilibrium model proposed by A. Zlotnick (J. Mol. Biol. 241:59-67, 1994).

Bacillus Phages↗
PubMed2024

Whole transcriptome sequencing analyses of islets reveal ncRNA regulatory networks underlying impaired insulin secretion and increased β-cell mass in high fat diet-induced diabetes mellitus.

AIM: Our study aims to identify novel non-coding RNA-mRNA regulatory networks associated with β-cell dysfunction and compensatory responses in obesity-related diabetes. METHODS: Glucose metabolism, islet architecture and secretion, and insulin sensitivity were characterized in C57BL/6J mice fed on a 60% high-fat diet (HFD) or control for 24 weeks. Islets were isolated for whole transcriptome sequencing to identify differentially expressed (DE) mRNAs, miRNAs, IncRNAs, and circRNAs. Regulatory networks involving miRNA-mRNA, lncRNA-mRNA, and lncRNA-miRNA-mRNA were constructed and functions were assessed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. RESULTS: Despite compensatory hyperinsulinemia and a significant increase in β-cell mass with a slow rate of proliferation, HFD mice exhibited impaired glucose tolerance. In isolated islets, insulin secretion in response to glucose and palmitic acid deteriorated after 24 weeks of HFD. Whole transcriptomic sequencing identified a total of 1324 DE mRNAs, 14 DE miRNAs, 179 DE lncRNAs, and 680 DE circRNAs. Our transcriptomic dataset unveiled several core regulatory axes involved in the impaired insulin secretion in HFD mice, such as miR-6948-5p/Cacna1c, miR-6964-3p/Cacna1b, miR-3572-5p/Hk2, miR-3572-5p/Cckar and miR-677-5p/Camk2d. Additionally, proliferative and apoptotic targets, including miR-216a-3p/FKBP5, miR-670-3p/Foxo3, miR-677-5p/RIPK1, miR-802-3p/Smad2 and ENSMUST00000176781/Caspase9 possibly contribute to the increased β-cell mass in HFD islets. Furthermore, competing endogenous RNAs (ceRNA) regulatory network involving 7 DE miRNAs, 15 DE lncRNAs and 38 DE mRNAs might also participate in the development of HFD-induced diabetes. CONCLUSIONS: The comprehensive whole transcriptomic sequencing revealed novel non-coding RNA-mRNA regulatory networks associated with impaired insulin secretion and increased β-cell mass in obesity-related diabetes.

Mice↗
PubMed1995

Colocalization of neuropeptide Y with other neurochemical markers in the guinea-pig small intestine.

The chemical coding and projections of neurons containing neuropeptide Y (NPY) have been investigated in the myenteric plexus of the guinea-pig small intestine. Chemical coding was determined by investigating the colocalization of NPY immunoreactivity with the immunoreactivities for bombesin (BN), 5-HT, nitric oxide synthase (NOS), and somatostatin. Projections were determined by studying the consequences of nerve lesions created by myectomy and myotomy operations. NPY immunoreactivity was localized in four classes of myenteric neuron, anally projecting interneurons, neurons that projected anally and to the circular muscle, neurons projecting to the longitudinal muscle and in a small population of secretomotor neurons that projected to the mucosa. The interneurons and muscle motor neurons both had Dogiel type I morphology, whereas the secretomotor neurons had fine branching processes. Of the NPY-immunoreactive Dogiel type I neurons, 98% were also immunoreactive for NOS; conversely, 82% of NOS-immunoreactive neurons were immunoreactive for NPY. BN was also colocalized with NPY and NOS; 30% of the NPY-immunoreactive neurons were BN/NOS/NPY-immunoreactive. No nerve cells had BN and NPY immunoreactivity without NOS immunoreactivity. The presence of NPY immunoreactivity was investigated in three classes of descending interneurons that are distinguished by their reactivities for somatostatin, 5-HT and NOS. NPY immunoreactivity was never colocalized with 5-HT or somatostatin, but most NPY-immunoreactive descending interneurons whose terminals formed pericellular baskets were also reactive for BN and NOS. The average projection lengths of the NPY interneurons was 2-3 mm, in the anal direction. Evaluation of immunoreactivity for BN, NOS and NPY revealed three major populations of anally directed circular muscle motor neurons, with BN/NOS/NPY, BN/NOS and NOS/NPY immunoreactivities. Examination of simultaneous labeling after nerve lesions showed that NOS/NPY neurons had short anal projections, averaging about 2-3 mm, and neurons with BN immunoreactivity were longer, having average projections of about 5-8 mm.

Animals↗
PubMed1993

Putative full-length clones of the genomic DNA segments of subterranean clover stunt virus and identification of the segment coding for the viral coat protein.

Subterranean clover stunt disease is an economically important aphid-borne virus disease affecting certain pasture and grain legumes in Australia. The virus associated with the disease, subterranean clover stunt virus (SCSV), was previously found to be representative of a new type of single-stranded DNA virus. Analysis of the virion DNA and restriction mapping of double-stranded cDNA synthesized from virion DNA suggested that SCSV has a segmented genome composed of 3 or 4 different species of circular ssDNA each of about 850-880 nucleotides. To further investigate the complexity of the SCSV genome, we have isolated the replicative form DNA from infected pea and from it prepared putative full-length clones representing the SCSV genome segments. Analysis of these clones by restriction mapping indicated that clones representing at least 4 distinct genomic segments were obtained. This method is thus suitable for generating an extensive genomic library of novel ssDNA viruses containing multiple genome segments such as SCSV and banana bunchy top virus. The N-terminal amino acid sequence and amino acid composition of the coat protein of SCSV were determined. Comparison of the amino acid sequence with partial DNA sequence data, and the distinctly different restriction maps obtained for the full-length clones suggested that only one of these clones contained the coat protein gene. The results confirmed that SCSV has a functionally divided genome composed of several distinct ssDNA circles each of about 1 kb.

Amino Acid Sequence↗
PubMed1996

Chemical coding of neurons in the myenteric plexus and external muscle of the small and large intestine of the mouse.

Immunohistochemical techniques were used to examine the presence and co-localisation of a range of putative neurotransmitters and other neuronal markers in the myenteric plexus of the small and large intestine of the mouse. Distinct sub-populations of myenteric neurons were identified, based on the combinations of substances they contained and the distribution of their fibres. In the small intestine, there were two major classes of circular muscle motor neurons; one class was characterised by the presence of nitric oxide synthase, vasoactive intestinal peptide plus neuropeptide Y (NOS/VIP/NPY), and the second class contained calretinin plus substance P (CalR/SP). There were seven classes of neurons that innervated myenteric ganglia; these contained nos, vip, nos/vip, npy, calr/calbindin (calb), sp or 5-ht. In the large intestine, there were five major classes of motor neurons that contained nos, nos/vip, gaba, sp, or calr/sp, and seven major classes of neurons that innervated myenteric ganglia and contained nos, vip, calr/calb, calr, sp, gaba or 5-ht. Although some aspects of the patterns of co-localisation are similar to those in other species, this study re-inforces recent analyses that indicate significant species differences in neurochemical patterns in the enteric neurons of different species.

Animals↗
PubMed1985

Gene disruption by transformation in Neurospora crassa.

To establish conditions which might permit deliberate gene disruptions in Neurospora crassa, we studied transformation with linear DNA fragments. The transformation frequency observed was increased about twofold in comparison with that obtained with circular plasmid DNA. However, only a low proportion, approximately 10%, of the integration events occurred at the homologous site, whereas most integrations of transforming DNA took place in nonhomologous regions. It was also found that multiple integration events frequently occurred in individual transformants. A plasmid, designated pJP12, was constructed that contains the N. crassa am+ gene interrupted by insertion into its coding region of a DNA segment carrying a functional Neurospora qa-2+ gene. A fragment of Neurospora DNA that contains this am qa-2+ construction was obtained from plasmid pJP12 and used to transform an am+ qa-2 strain in an attempt to disrupt the resident am+ gene. After the initial qa-2+ transformants were converted to homokaryons by appropriate crosses, 10 independent transformants with an am mutant phenotype were found among 117 examined. Each of these qa-2+ am transformants showed the loss of a hybridization band in Southern blots of genomic DNA that corresponded to the normal am+ gene and the presence of a new hybridization band, consistent with an alteration in the am+ region.

DNA, Fungal↗
PubMed1987

The nucleotide sequence of a 3.2 kb segment of mitochondrial maxicircle DNA from Crithidia fasciculata containing the gene for cytochrome oxidase subunit III, the N-terminal part of the apocytochrome b gene and a possible frameshift gene; further evidence for the use of unusual initiator triplets in trypanosome mitochondria.

A 3.2 kb segment of the maxicircle of Crithidia fasciculata mitochondrial (mt) DNA contains the gene for cytochrome oxidase subunit III (coxIII), the N-terminal portion of the gene for apocytochrome b (cytb) and two partially overlapping Unassigned Reading Frames (C.URF2/1). Transcript analysis of the segment reveals that both the coxIII gene and the C.URF2/1 area are transcribed into a pair of RNA products. With the C. fasciculata gene version as a probe, a coxIII gene could not be detected in nuclear and mtDNA of Trypanosoma brucei, indicating that the cytochrome oxidases of these two closely related trypanosome species may differ. The nucleotide homology in the N-terminal region of the coxIII and cytb genes in T. brucei, Leishmania tarentolae and C. fasciculata starts at a UUA leucine codon, which adds further support to the hypothesis that apart from AUG, other initiator triplets are used in trypanosomal mitochondria: UUG, CUG and UUA, all triplets coding for leucine in the universal code. Finally, the possibility is discussed that the two overlapping URFs (C.URF2/1) in fact represent a single, frameshift containing, gene.

Amino Acid Sequence↗
PubMed1997

A cDNA clone to clover yellow vein potyvirus genome is highly infectious.

We obtained a highly infectious cDNA clone of clover yellow vein virus (CIYVV). The cDNA fragments, from which a full-length cDNA clone was constructed, were sequenced, and the complete nucleotide sequence of C1YVV RNA was determined. The viral genome is 9584 nucleotides (nt) in length excluding the poly(A) tail and contains one open reading frame (ORF) encoding a large polyprotein of 3072 amino acids. The non-coding region preceding the ORF is 190 nt long. The termination codon is followed by a 175-nt sequence. Seven potential protease NIa, one HC-pro and one P1 protease recognition sites were found in the C1YVV polyprotein by searching for cleavage consensus sequences among the potyvirus group. The cleavage dipeptides of C1YVV NIa protease are Q(E)/S(A,G). The F is conserved at the -2 position from the cleavage site except for at the P3/6K1 junction, and the V conserved at the -4 position among many potyviruses is not present at all. The genome organization of C1YVV was determined, and the amino acid sequence was compared with that of other potyviruses. The full-length cDNA clone of C1YVV was constructed by combining cDNA fragments and placed it under the control of the cauliflower mosaic virus 35S promoter. The full-length cDNA was constructed so that no extra nucleotide was present at the transcription initiation site and only 10 adenine residues were present at the 3' end of the C1YVV cDNA clone. Mechanical inoculation of a circular-formed plasmid DNA onto broad bean seedlings led to systemic infection, and the symptoms were similar to those caused by the wild-type virus but rather mild. Plasmid diluted as low as 500 pg/microl was able to induce symptoms, demonstrating that this full-length C1YVV cDNA is more infectious than any other infectious cDNAs so far reported. Filamentous particles reacting with the antiserum to C1YVV were observed in the crude sap of infected plants by immunoelectron microscopy, and genome replication was demonstrated by RT-PCR of 3' non-coding regions of C1YVV genome in total plant RNAs.

Amino Acid Sequence↗
PubMed1985

Severe idiopathic constipation is associated with a distinctive abnormality of the colonic myenteric plexus.

We analyzed the clinical, radiographic, esophageal manometric, and pathologic features of 26 women with severe, idiopathic constipation. Twenty-four patients were between 19 and 39 yr of age. Stool frequency was once every 5-28 days. On barium enema examination, 9 of 24 patients had colons of increased length and 4 of these 9 patients had colons of increased width (greater than 10 cm). Radionuclide solid-meal gastric-emptying studies were normal in 23 patients tested. Esophageal manometry demonstrated high-amplitude waves in 10 of 22 patients and long-duration waves in 3 of these 10 patients. Rectal biopsy specimens showed normal submucosal neurons in all patients and melanosis coli in 6. Twelve patients underwent subtotal colectomies for constipation. Conventional light microscopy using hematoxylin and eosin serial sections showed (a) melanosis coli in 4 patients; (b) normal smooth muscle in 11; (c) thinning of the circular muscle in 1; and (d) no apparent abnormalities of the myenteric plexus in any. In contrast, silver stains of the myenteric plexus showed (a) quantitatively reduced numbers of argyrophilic neurons in 10 patients; (b) morphologically abnormal argyrophilic neurons in 11; (c) decreased numbers of axons in 11; and (d) increased numbers of variably sized nuclei within ganglia in all 12. A coded analysis of the silver stains of colons from 8 patients with constipation and 19 control cases demonstrated that the pathologic abnormalities of severe idiopathic constipation could be differentiated from controls. Thus, severe idiopathic constipation is associated with a pathologically identifiable abnormality of the myenteric plexus. This abnormality appears different from anything previously described in intestinal pseudoobstruction.

Adult↗