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At least 325 records · Page 18Linked to original sources
PubMed1998

The C-terminal region of the Escherichia coli UvrC protein, which is homologous to the C-terminal region of the human ERCC1 protein, is involved in DNA binding and 5'-incision.

The incisions in the DNA at the 3'- and 5'-side of a DNA damage during nucleotide excision repair in Escherichia coli occur in a complex consisting of damaged DNA, UvrB and UvrC. The exact requirements for the two incision events, however, are different. It has previously been shown that the 3'-incision requires the interaction between the C-terminal domain of UvrB and a homologous region in UvrC. This interaction, however, is dispensable for the 5'-incision. Here we show that the C-terminal domain of the UvrC protein is essential for the 5'-incision, whereas this domain can be deleted without affecting the 3'-incision. The C-terminal domain of UvrC is homologous with the C-terminal part of the ERCC1 protein which, in a complex with XPF, is responsible for the 5'-incision reaction in human nucleotide excision repair. Both in the UvrC and the ERCC1 domain a Helix-hairpin-Helix (HhH) motif can be indicated, albeit at different positions. Such a motif also has been found in a large variety of DNA binding proteins and it has been suggested to form a structure involved in non-sequence-specific DNA binding. In contrast to the full length UvrC protein, a truncated UvrC protein (UvrC554) lacking the entire ERCC1 homology including the HhH motif no longer binds to ssDNA. Analysis of protein-DNA complexes using bandshift experiments showed that this putative DNA binding domain of UvrC is required for stabilisation of the UvrBC-DNA complex after the 3'-incision has taken place. We propose that after the initial 3'-incision the HhH motif recognises a specific DNA structure, thereby positioning the catalytic site for the subsequent 5'-incision reaction.

Amino Acid Sequence↗
PubMed1981

The 3'-terminal region of bacterial 23S ribosomal RNA: structure and homology with the 3'-terminal region of eukaryotic 28S rRNA and with chloroplast 4.5s rRNA.

The sequence of the 110 nucleotide fragment located at the 3'-end of E.coli, P.vulgaris and A.punctata 23S rRNAs has been determined. The homology between the E.coli and P.vulgaris fragments is 90%, whereas that between the E.coli and A.punctate fragments is only 60%. The three rRNA fragments have sequences compatible with a secondary structure consisting of two hairpins. Using chemical and enzymatic methods recently developed for the study of the secondary structure of RNA, we demonstrated that one of these hairpins and part of the other are actually present in the three 3'-terminal fragments in solution. This supports the existence of these two hairpins in the intact molecule. Indeed, results obtained upon limited digestion of intact 23S RNA with T1 RNase were in good agreement with the existence of these two hairpins. We observed that the primary structures of the 3'-terminal regions of yeast 26S rRNA and X.laevis 28S rRNA are both compatible with a secondary structure similar to that found at the 3'-end of bacterial 23S rRNAs. Furthermore, both tobacco and wheat chloroplast 4.5S rRNAs can also be folded in a similar way as the 3'-terminal region of bacterial 23S rRNA, the 3'-end of chloroplast 4.5S rRNAs being complementary to the 5'-end of chloroplast 23S rRNA. This strongly reinforces the hypothesis that chloroplast 4.5S rRNA originates from the 3'-end of bacterial 23S rRNA and suggests that this rRNA may be base-paired with the 5'-end of chloroplast 23S rRNA. Invariant oligonucleotides are present at identical positions in the homologous secondary structures of E.coli 23S, yeast 26S, X.laevis 28S and wheat and tobacco 4.5S rRNAs. Surprisingly, the sequences of these oligonucleotides are not all conserved in the 3'-terminal regions of A.punctata or even P.vulgaris 23S rRNAs. Results obtained upon mild methylation of E.coli 50S subunits with dimethylsulfate strongly suggest that these invariant oligonucleotides are involved in RNA tertiary structure or in RNA-protein interactions.

Animals↗
PubMed2000

Distinctive functions of Syk N-terminal and C-terminal SH2 domains in the signaling cascade elicited by oxidative stress in B cells.

Syk plays a crucial role in the transduction of oxidative stress signaling. In this paper, we investigated the roles of Src homology 2 (SH2) domains of Syk in oxidative stress signaling, using Syk-negative DT40 cells expressing the N- or C-terminal SH2 domain mutant [mSH2(N) or mSH2(C)] of Syk. Tyrosine phosphorylation of Syk in cells expressing mSH2(N) Syk after H(2)O(2) treatment was higher than that in cells expressing wild-type Syk or mSH2(C) Syk. The tyrosine phosphorylation of wild-type Syk and mSH2(C) Syk, but not that of mSH2(N), was sensitive to PP2, a specific inhibitor of Src-family protein-tyrosine kinase. In oxidative stress, the C-terminal SH2 domain of Syk was demonstrated to be required for induction of tyrosine phosphorylation of cellular proteins, phospholipase C (PLC)-gamma2 phosphorylation, inositol 1,4, 5-triphosphate (IP(3)) generation, Ca(2)(+) release from intracellular stores, and c-Jun N-terminal kinase activation. In contrast, in mSH2(N) Syk-expressing cells, tyrosine phosphorylation of intracellular proteins including PLC-gamma2 was markedly induced in oxidative stress. The enhanced phosphorylation of mSH2(N) Syk and PLC-gamma2, however, did not link to Ca(2)(+) mobilization from intracellular pools and IP(3) generation. Thus, the N- and C-terminal SH2 domains of Syk possess distinctive functions in oxidative stress signaling.

Animals↗
PubMed1993

Molecular conformation of porcine amelogenin in solution: three folding units at the N-terminal, central, and C-terminal regions.

Circular dichroism (CD) studies were conducted to gain a better insight into the conformation of amelogenins, which were isolated from developing enamel of piglets. The intact porcine amelogenin and its degraded products were purified chromatographically. The 25-residue peptide corresponding to the segment at the C-terminus was synthesized. CD spectra of these samples were measured at pH 5.0-5.3 in the temperature range between 4 and 90 degrees C. The most remarkable finding was that the CD spectrum of the intact amelogenin was accounted for by the sum of the spectra of the three fragments at the N-terminal, central, and C-terminal regions, supporting the hypothesis that the structure of the whole protein consists of discrete folding units. Furthermore, low-angle laser light scattering analysis provided evidence that the 20 kDa amelogenin, the most abundant extracellular matrix protein in forming enamel tissue, exists in a monomeric form at pH 5.3 and 25 degrees C. It was tentatively concluded that the N-terminal region contains beta-sheet structures, while the spectral characteristics of the C-terminal region are similar to those of a random coil conformation. The conformation of the central region was characterized by a strong negative ellipticity at 203 nm, although its nature remains to be defined.

Amelogenin↗
PubMed2005

The intrinsically disordered C-terminal domain of the measles virus nucleoprotein interacts with the C-terminal domain of the phosphoprotein via two distinct sites and remains predominantly unfolded.

Measles virus is a negative-sense, single-stranded RNA virus within the Mononegavirales order,which includes several human pathogens, including rabies, Ebola, Nipah, and Hendra viruses. The measles virus nucleoprotein consists of a structured N-terminal domain, and of an intrinsically disordered C-terminal domain, N(TAIL) (aa 401-525), which undergoes induced folding in the presence of the C-terminal domain (XD, aa 459-507) of the viral phosphoprotein. With in N(TAIL), an alpha-helical molecular recognition element (alpha-MoRE, aa 488-499) involved in binding to P and in induced folding was identified and then observed in the crystal structure of XD. Using small-angle X-ray scattering, we have derived a low-resolution structural model of the complex between XD and N(TAIL), which shows that most of N(TAIL) remains disordered in the complex despite P-induced folding within the alpha-MoRE. The model consists of an extended shape accommodating the multiple conformations adopted by the disordered N-terminal region of N(TAIL), and of a bulky globular region, corresponding to XD and to the C terminus of N(TAIL) (aa 486-525). Using surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and heteronuclear magnetic resonance, we show that N(TAIL) has an additional site (aa 517-525) involved in binding to XD but not in the unstructured-to-structured transition. This work provides evidence that intrinsically disordered domains can establish complex interactions with their partners, and can contact them through multiple sites that do not all necessarily gain regular secondary structure.

Binding Sites↗
PubMed2000

Screening of BRCA1 mutation using immunohistochemical staining with C-terminal and N-terminal antibodies in familial ovarian cancers.

We examined the subcellular localization of BRCA1 proteins using immunohistochemical staining with C-terminal (GLK-2 antibody) and N-terminal (Ab-2 antibody) monoclonal antibodies in 44 familial ovarian cancers. Among these, 24 cases were associated with 13 independent germ-line mutations of BRCA1, and loss of heterozygosity (LOH) at one or more BRCA1 microsatellite markers was found in all 21 informative tumors tested. With GLK-2 antibody, cytoplasmic staining was observed in 15 of 16 tumors (93.8%) with mutation in exon 11, and BRCA1 staining was absent in 8 of 8 tumors (100%) with mutation in exons other than exon 11. When immunohistochemical staining was performed with Ab-2 antibody, both nuclear and cytoplasmic staining were observed in 14 of 16 tumors (87.5%) with mutation in exon 11. Interestingly, nuclear staining was observed in 3 of 3 tumors (100%) with mutation downstream of exon 11, even though no staining was detected in 5 of 5 tumors (100%) with mutation upstream of exon 11. On the other hand, in familial ovarian cancers without BRCA1 mutations, nuclear staining or both nuclear and cytoplasmic staining was observed in 18 of 20 specimens (90%) and 20 of 20 specimens (100%) with GLK-2 antibody and with Ab-2 antibody, respectively. These results suggest that an immunohistochemical assay in combination with employing the C-terminal and the N-terminal antibodies appears to have potential as a reliable and useful technique for the screening of BRCA1 mutations, at least to predict the status of mutation, upstream or downstream of exon 11.

BRCA1 Protein↗
PubMed1992

Substance P-induced granulocyte infiltration in mouse skin: the mast cell-dependent granulocyte infiltration by the N-terminal peptide is enhanced by the activation of vascular endothelial cells by the C-terminal peptide.

Previous studies have shown that substance P induces granulocyte infiltration in mouse skin, which is mediated through mast cell degranulation. However, it is not yet known whether the direct effect of substance P on vascular endothelial cells is involved in the granulocyte infiltration in the skin. To solve this issue, we used the N-terminal peptide substance P1-9 (SP1-9), which is active for mast cells but inactive for vascular endothelial cells, and the C-terminal peptide SP6-11, which is active for vascular endothelial cells but inactive for mast cells, since substance P activates both mast cells and vascular endothelial cells. The subcutaneous administration of substance P (10(-7)-10(-5)M) caused granulocyte (neutrophil and eosinophil) infiltration in the skin of BALB/c mice 6 h after the injection. SP1-9 (10(-5)-10(-4) M) also caused granulocyte infiltration of mouse skin which was associated with mast cell degranulation. In contrast, SP6-11 (10(-7)-10(-4) M), which was found to increase the vascular permeability of endothelial cells in mouse skin, induced no significant granulocyte infiltration nor mast cell degranulation. However, SP6-11 (10(-5)-10(-4) M) enhanced SP1-9-induced granulocyte infiltration in the skin without any significant increase in mast cell degranulation. We conclude that substance P causes granulocyte infiltration in mouse skin through both mast cell degranulation induced by the N-terminal peptide of substance P and the activation of vascular endothelial cells induced by the C-terminal peptide of substance P.

Animals↗
PubMed2005

LEAFY, TERMINAL FLOWER1 and AGAMOUS are functionally conserved but do not regulate terminal flowering and floral determinacy in Impatiens balsamina.

In Impatiens balsamina a lack of commitment of the meristem during floral development leads to the continuous requirement for a leaf-derived floral signal. In the absence of this signal the meristem reverts to leaf production. Current models for Arabidopsis state that LEAFY (LFY) is central to the integration of floral signals and regulates flowering partly via interactions with TERMINAL FLOWER1 (TFL1) and AGAMOUS (AG). Here we describe Impatiens homologues of LFY, TFL1 and AG (IbLFY, IbTFL1 and IbAG) that are highly conserved at a sequence level and demonstrate homologous functions when expressed ectopically in transgenic Arabidopsis. We relate the expression patterns of IbTFL1 and IbAG to the control of terminal flowering and floral determinacy in Impatiens. IbTFL1 is involved in controlling the phase of the axillary meristems and is expressed in axillary shoots and axillary meristems which produce inflorescences, but not in axillary flowers. It is not involved in maintaining the terminal meristem in either an inflorescence or indeterminate state. Terminal flowering in Impatiens appears therefore to be controlled by a pathway that uses a different integration system than that regulating the development of axillary flowers and branches. The pattern of ovule production in Impatiens requires the meristem to be maintained after the production of carpels. Consistent with this morphological feature IbAG appears to specify stamen and carpel identity, but is not sufficient to specify meristem determinacy in Impatiens.

AGAMOUS Protein, Arabidopsis↗
PubMed1975

Sequence determination of 5'-terminal and 3'-terminal T1 oligonucleotides of 18-S ribosomal RNA of a mouse cell line (L 5178 Y).

The 5' and 3'-terminal oligonucleotides of 18-S ribosomal RNA of L 5178 Y (a mouse cell line) obtained after total T1 ribonuclease hydrolysis were isolated by a diagonal procedure. They were localized on the fingerprint of T1-ribonuclease-hydrolysed 18-S RNA. These two oligonucleotides were partially hydrolysed by snake venom and spleen phsophodiesterases and resulting products were fractionated bidimensionally. Their base compositions were determined by total hydrolysis with piperidine or snake venom phosphodiesterase. From these results the following sequences were deduced: pU-A-C-C-U-G for the 5'-terminal oligonucleotide and G-A-U-C-A-U-U-Aoh for the 3'-terminal oligonucleotide. Quantitative studies indicated that these sequences represent at least 70% for the 5' oligonucleotide and 85% for the 3' oligonucleotide of the terminal sequences of the 18-S RNA.

Animals↗
PubMed1988

The primary structure of the histone H2A(2) type from wheat germ. A core histone type with both, N-terminal and C-terminal extensions.

The histone H2A(2) type from wheat germ comprises at least two highly homologous isohistones with 151 amino acid residues. Microheterogeneity occurs mainly at the N-terminal and C-terminal regions. These isohistones have both N-terminal (7 amino acid residues) and C-terminal (15 amino acid residues) extensions relative to calf thymus histone H2A.

Amino Acid Sequence↗
PubMed1997

The catalytic subunit of Dictyostelium cAMP-dependent protein kinase -- role of the N-terminal domain and of the C-terminal residues in catalytic activity and stability.

The C subunit of Dictyostelium cAMP-dependent protein kinase (PKA) is unusually large (73 kDa) due to the presence of 330 amino acids N-terminal to the conserved catalytic core. The sequence following the core, including a C-terminal -Phe-Xaa-Xaa-Phe-COOH motif, is highly conserved. We have characterized the catalytic activity and stability of C subunits mutated in sequences outside the catalytic core and we have analyzed their ability to interact with the R subunit and with the heat-stable protein-kinase inhibitor PKI. Mutants carrying deletions in the N-terminal domain displayed little difference in their kinetic properties and retained their capacity to be inhibited by R subunit and by PKI. In contrast, the mutation of one or both of the phenylalanine residues in the C-terminal motif resulted in a decrease of catalytic activity and stability of the proteins. Inhibition by the R subunit or by PKI were however unaffected. Sequence-comparison analysis of other protein kinases revealed that a -Phe-Xaa-Xaa-Phe- motif is present in many Ser/Thr protein kinases, although its location at the very end of the polypeptide is a particular feature of the PKA family. We propose that the presence of this motif may serve to identify isoforms of protein kinases.

Amino Acid Sequence↗
PubMed1995

The effect of opiates on the terminal nerve impulse and quantal secretion from visualized amphibian nerve terminals.

1. Secretion of transmitter from amphibian motor nerve terminal release sites is intermittent, spatially non-uniform and varies considerably throughout the year and during development. The role of opioid receptors in modulating transmitter secretion from amphibian motor nerve terminals is evaluated in this study. 2. Dynorphin-A (24 microM) and morphine (500 microM) did not significantly change the shape of the nerve impulse or the consistency with which it was observed, but decreased evoked quantal secretion by more than 50%. These effects of dynorphin-A and morphine were largely reversed by naloxone (50 microM). 3. Dynorphin-A and morphine did not significantly change either the amplitude or the frequency of spontaneous quantal secretions. 4. There was a uniform decrease in evoked quantal secretion from release sites along terminal branches, irrespective of the quantal content value before drug treatment, indicating no difference in the susceptibility of proximal vs distal release sites to opiates. 5. Increasing the extracellular calcium concentration (0.3 to 0.4 mM) or trains of conditioning-test impulses (25 to 100 Hz) resulted in smaller dynorphin-A or morphine-induced decreases in evoked quantal secretion. 6. The decrease in evoked quantal secretion occurs as a result of a uniform decrease in the probability of quantal secretion from release sites without any affect on the propagation of the nerve terminal impulse. Low probability release sites become effectively silent.

Action Potentials↗
PubMed2005

Structural function of C-terminal amidation of endomorphin. Conformational comparison of mu-selective endomorphin-2 with its C-terminal free acid, studied by 1H-NMR spectroscopy, molecular calculation, and X-ray crystallography.

To investigate the structural function of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH(2)), an endogenous micro-opioid receptor ligand, the solution conformations of EM2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH) in TFE (trifluoroethanol), water (pH 2.7 and 5.2), and aqueous DPC (dodecylphosphocholine) micelles (pH 3.5 and 5.2) were investigated by the combination of 2D (1)H-NMR measurement and molecular modelling calculation. Both peptides were in equilibrium between the cis and trans rotamers around the Tyr--Pro w bond with population ratios of 1 : 1 to 1 : 2 in dimethyl sulfoxide, TFE and water, whereas they predominantly took the trans rotamer in DPC micelle, except in EM2OH at pH 5.2, which had a trans/cis rotamer ratio of 2 : 1. Fifty possible 3D conformers were generated for each peptide, taking different electronic states depending on the type of solvent and pH (neutral and monocationic forms for EM2, and zwitterionic and monocation forms for EM2OH) by the dynamical simulated annealing method, under the proton-proton distance constraints derived from the ROE cross-peak intensities. These conformers were then roughly classified into four groups of two open [reverse S (rS)- and numerical 7 (n7)-type] and two folded (F1- and F2-type) conformers according to the conformational pattern of the backbone structure. Most EM2 conformers in neutral (in TFE) and monocationic (in water and DPC micelles) forms adopted the open structure (mixture of major rS-type and minor n7-type conformers) despite the trans/cis rotamer form. On the other hand, the zwitterionic EM2OH in TFE, water and DPC micelles showed an increased population of F1- and F2-type folded conformers, the population of which varied depending on their electronic state and pH. Most of these folded conformers took an F1-type structure similar to that stabilized by an intramolecular hydrogen bond of (Tyr1)NH(3) (+)...COO(-)(Phe4), observed in its crystal structure. These results show that the substitution of a carboxyl group for the C-terminal amide group makes the peptide structure more flexible and leads to the ensemble of folded and open conformers. The conformational requirement of EM2 for binding to the micro-opioid receptor and the structural function of the C-terminal amide group are discussed on the basis of the present conformational features of EM2 and EM2OH and a possible model for binding to the micro-opioid receptor, constructed from the template structure of rhodopsin.

Amides↗
PubMed1994

Cloning and nucleotide sequence of type 3 M protein gene (emm3) consisting of an N-terminal variable portion and C-terminal conserved C repeat regions: relation to other genes of Streptococcus pyogenes. Streptococcal Diseases Study Group.

The structural gene for type 3 M protein of Streptococcus pyogenes, which consists of an N-terminal variable portion and C-terminal conserved repeat regions, has been cloned by the polymerase chain reaction (PCR) with two primers (K-1 and K-2). They were selected from the best conserved region of the leader sequences and of the C-terminal portion near the Hexapeptide (LPSTGE) sequence found in different M proteins. From the nucleotide sequence of the product, 1645 base pairs were determined, including 32 amino acids of the leader sequences, the complete N-terminal variable region and the conserved C repeat region. Analysis of the deduced amino acids of the sequence revealed the existence of two major repeat regions, the B and C repeat regions. Comparison of the C-repeat regions among M3 and other M proteins showed them to be more than 90% identical. The two B repeat blocks in M3 protein are also similar to those in M12 protein. Predictive secondary structure analysis of M3 protein reveals a strong alpha-helical potential. The algorithm also shows that the beta-sheet and turn potential for region 23-42 in M3 protein are similar to those for region 28-50 in M12 protein. The results indicate that M3 protein is closely related to M12 protein.

Amino Acid Sequence↗
PubMed2003

Formation of pseudo-terminal restriction fragments, a PCR-related bias affecting terminal restriction fragment length polymorphism analysis of microbial community structure.

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called "pseudo-T-RFs" since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.

Animals↗
PubMed1999

Role of TolR N-terminal, central, and C-terminal domains in dimerization and interaction with TolA and tolQ.

The Tol-PAL system of Escherichia coli is a multiprotein system involved in maintaining the cell envelope integrity and is necessary for the import of some colicins and phage DNA into the bacterium. It is organized into two complexes, one near the outer membrane between TolB and PAL and one in the cytoplasmic membrane between TolA, TolQ, and TolR. In the cytoplasmic membrane, all of the Tol proteins have been shown to interact with each other. Cross-linking experiments have shown that the TolA transmembrane domain interacts with TolQ and TolR. Suppressor mutant analyses have localized the TolQ-TolA interaction to the first transmembrane domain of TolQ and have shown that the third transmembrane domain of TolQ interacts with the transmembrane domain of TolR. To get insights on the composition of the cytoplasmic membrane complex and its possible contacts with the outer membrane complex, we focused our attention on TolR. Cross-linking and immunoprecipitation experiments allowed the identification of Tol proteins interacting with TolR. The interactions of TolR with TolA and TolQ were confirmed, TolR was shown to dimerize, and the resulting dimer was shown to interact with TolQ. Deletion mutants of TolR were constructed, and they allowed us to determine the TolR domains involved in each interaction. The TolR transmembrane domain was shown to be involved in the TolA-TolR and TolQ-TolR interactions, while TolR central and C-terminal domains appeared to be involved in TolR dimerization. The role of the TolR C-terminal domain in the TolA-TolR interaction and its association with the membranes was also demonstrated. Furthermore, phenotypic studies clearly showed that the three TolR domains (N terminal, central, and C terminal) and the level of TolR production are important for colicin A import and for the maintenance of cell envelope integrity.

Bacterial Proteins↗
PubMed1983

A unique sequence in murine leukemia virus long terminal repeat functions as a termination signal for transcription in Escherichia coli.

Moloney murine leukemia virus DNA fragments were subcloned into two plasmids downstream of active promoters to detect termination sequences for transcription initiated at these promoters. Two of the viral DNA fragments (2,050 and 595 base pairs) were able to block transcription when inserted in one orientation but not when inserted in the other. These two viral DNA fragments contained long terminal repeat sequences. Deletion of 84 base pairs from the U-3 and R regions in the long terminal repeat removed the transcription termination activity.

Cloning, Molecular↗
PubMed1998

Duck hepatitis B virus nucleocapsids formed by N-terminally extended or C-terminally truncated core proteins disintegrate during viral DNA maturation.

Hepadnaviruses are DNA viruses that replicate through reverse transcription of an RNA pregenome. Viral DNA synthesis takes place inside viral nucleocapsids, formed by core protein dimers. Previous studies have identified carboxy-terminal truncations of the core protein that affect viral DNA maturation. Here, we describe the effect of small amino-terminal insertions into the duck hepatitis B virus (DHBV) core protein on viral DNA replication. All insertion mutants formed replication-competent nucleocapsids. Elongation of viral DNA, however, appeared to be incomplete. Increasing the number of additional amino acids and introducing negatively charged residues further reduced the observed size of mature viral DNA species. Mutant core proteins did not inhibit the viral polymerase. Instead, viral DNA synthesis destabilized mutant nucleocapsids, rendering mature viral DNA selectively sensitive to nuclease action. Interestingly, the phenotype of two previously described carboxy-terminal DHBV core protein deletion mutants was found to be based on the same mechanism. These data suggest that (i) the amino- as well as the carboxy-terminal portion of the DHBV core protein plays a critical role in nucleocapsid stabilization, and (ii) the hepadnavirus polymerase can perform partial second-strand DNA synthesis in the absence of intact viral nucleocapsids.

Amino Acid Sequence↗