Analysis of 5' non-coding region in hepatitis C virus by single-strand conformation polymorphism and low-stringency single specific primer PCR.

Single-strand conformation polymorphism (SSCP) and low-stringency single specific primer (LSSP)-PCR in hepatitis C virus (HCV) genotyping were examined for informativeness and reliability. The analysis of HCV isolates included seven type 1 isolates, two type 2 isolates, and two type 3 isolates. We also analyzed five isolates that presented as mixed infections determined by type-specific PCR. Among mixed isolates, one isolate was 1a/1b and four isolates were 1b/3a. SSCP and LSSP-PCR were applied to the analysis of 5' non-coding region of HCV (-289 to -5) that contains genotype-specific sequences. Direct cycle sequencing of this region determined sequence divergences that define genotype and sequence alterations within the same genotype. Optimized conditions for the SSCP analysis clearly distinguished between genotypes 1, 2 and 3. In addition, the SSCP analysis detected sequence variants within the same genotype. However, the SSCP analysis and DNA sequencing did not confirm the presence of mixed infections. LSSP analysis, not previously employed in HCV genotyping, enabled clear distinction between genotypes 1, 2 and 3, however, this method did not differentiate between sequence variants within a genotype. Importantly, the LSSP profile demonstrated distinction between mixed infection isolates.

Human extracellular proteins display a different pattern of local sequence similarity with the four classes of human T-cell receptor V-regions than foreign proteins and human intracellular proteins: a preliminary report.

A pool of 110 randomly selected/generated amino acids sequences was used to perform specific local sequence similarity alignment analysis with the pool of 279 reported sequences of human T-cell receptor (TCR) V-regions. The 110 analyzed sequences were divided, according to their origin and nature, into six protein groups, as: human intracellular (hi), extracellular/transmembrane (he) and extracellular adhesive matrix (ha) proteins, 'average' human proteins (hum), proteins of non-human origin (nhum) and randomly generated quasi-protein sequences (r). These sequences were decomposed into all their overlapping 11-mer segments, generating a total of 56,836 derived peptides (at least 8000 per group). Each derived peptide was aligned with the 279 human TCR V-regions and assigned to the category (alpha-like, beta-like, gamma-like or delta-like) corresponding to the class (V alpha, Vbeta, Vgamma or Vdelta) of the V-region encompassing the most similar segment, as determined by the performed similarity-search. The six protein groups were found to differ significantly in their distribution of derived peptides among the four categories. According to the binomial tests results, human proteins from the extracellular compartment (he, ha) comprise a higher proportion of delta-like segments (P = 2.3 x 10(-2) and P < 10(-8), respectively) than the 'average' human proteins (hum). In addition, and in accordance with this finding, proteins that are normally not found in that topological compartment comprise a lower proportion of delta-like peptides (P = 1.4 x 10(-5) and P < 10(-8) for groups nhum and hi, respectively) than the 'average' human proteins (hum). In contrast, these proteins comprise a higher proportion of gamma-like segments (P = 8.3 x 10(-3), P = 1.4 x 10(-3) and P = 1.7 x 10(-4), for groups r, nhum and hi, respectively) than the 'average' human proteins (hum). These findings indicate significant differences between proteins encountered in the extracellular compartment--that are normally immunologically tolerated--and those the presence of which is usually non-tolerated. The results suggest that the discrimination and the reaction of the human immune network to proteins found in the extracellular compartment correlate with the proteins' pattern of preferential local sequence similarity with the Vgamma and Vdelta classes of human TCR V-regions, implying a specific and an important role of gammadelta-cells in the maintenance of the immune homeostasis. Whether this implication represents a rule associated with self-tolerance, will be investigated by future analyses.

Further evidence for the relationship of HIV-1 gp120 V3 loops with Ig superfamily members: similarity with the putative CDR3 region of T-cell receptor delta-chains.

Twenty-five V3 loops of envelope gp 120 extracted from 30 HIV-1 isolates were compared with T-cell receptor (TCR) subunits variable (V) portions using pairwise alignments of 11-residue peptides. The results indicate that, in comparison with random sequences, the analyzed V3 loops, unlike control (unrelated) sequences, display highly significant local similarity with TCR V delta (p approximately 10(-20)). However, pattern-matching searches were performed on a much larger number of V3 loops (484). In particular, selective pattern TR * * * NT * K * I is shared by V delta from human T-cell line KT19E and 230 HIV-1 V3 loops (N-terminal portion). Pattern RA * YT * * * I * G is common for V delta chain isolated from T-cell line DS6 of an immunodeficient patient and 69 V3 loops (C-terminal portion). The presented delta-chain portions of sequence similarity with the V3 loops overlap the putative complementarity-determining region (CDR3), thus possibly indicating functional similarity too.

The SBASE protein domain library, release 3.0: a collection of annotated protein sequence segments.

SBASE 3.0 is the third release of SBASE, a collection of annotated protein domain sequences. SBASE entries represent various structural, functional, ligand-binding and topogenic segments of proteins as defined by their publishing authors. SBASE can be used for establishing domain homologies using different database-search tools such as FASTA [Lipman and Pearson (1985) Science, 227, 1436-1441], and BLAST3 [Altschul and Lipman (1990) Proc. Natl. Acad. Sci. USA, 87, 5509-5513] which is especially useful in the case of loosely defined domain types for which efficient consensus patterns can not be established. The present release contains 41,749 entries provided with standardized names and cross-referenced to the major protein and nucleic acid databanks as well as to the PROSITE catalogue of protein sequence patterns. The entries are clustered into 2285 groups using the BLAST algorithm for computing similarity measures. SBASE 3.0 is freely available on request to the authors or by anonymous 'ftp' file transfer from < ftp.icgeb.trieste.it >. Individual records can be retrieved with the gopher server at < icgeb.trieste.it > and with a www-server at < http:@www.icgeb.trieste.it >. Automated searching of SBASE by BLAST can be carried out with the electronic mail server < sbase@icgeb.trieste.it >. Another mail server < domain@hubi.abc.hu > assigns SBASE domain homologies on the basis of SWISS-PROT searches. A comparison of pertinent search strategies is presented.

Immunoglobulin-like domain of HIV-1 envelope glycoprotein gp120 encodes putative internal image of some common human proteins.

By examining sequence similarity between the V3-loop of gp120 from various HIV-1 isolates and human proteins, we found that the V3 loop portion KKGIAIGPGR in strain New York 5 (HIV-1NY5) shares 70% identical residues with the collagen-like region (CLR) of human complement component C1q-A. C1q CLR was found to react with autoantibodies from several autoimmune disorders. Thus, we assumed that it would be of interest to find out the C1q reactivity with antibodies from AIDS sera. The results obtained show that the V3 loop-derived synthetic peptide KKGIAIGPGRTLY reacts both with AIDS patients sera and with antibodies purified on the V3 loop peptide-affinity column. The same affinity-purified antibodies bind also to C1q molecules. Since, according to our previous results, HIV-1 V3 loops and immunoglobulin heavy chain variable regions (Ig VH) share several common features, we suggest that the envelope of HIV-1NY5 bears a functional internal image of the C1q-A CLR epitope. Therefore, gp120 could manipulate the immune network and contribute to HIV-induced autoimmunity.

The SBASE protein domain library, release 2.0: a collection of annotated protein sequence segments.

SBASE 2.0 is the second release of SBASE, a collection of annotated protein domain sequences. SBASE entries represent various structural, functional, ligand-binding and topogenic segments of proteins [Pongor, S. et al. (1993) Prot. Eng., in press]. This release contains 34,518 entries provided with standardized names and it is cross-referenced to the major protein and nucleic acid databanks as well as to the PROSITE catalog of protein sequence patterns [Bairoch, A. (1992) Nucl. Acids Res., 20 suppl, 2013-2018]. SBASE can be used for establishing domain homologies using different database-search tools such as FASTA [Lipman and Pearson (1985) Science, 227, 1436-1441], FASTDB [Brutlag et al. (1990) Comp. Appl. Biosci., 6, 237-245] or BLAST3 [Altschul and Lipman (1990) Proc. Natl. Acad. Sci. USA, 87, 5509-5513] which is especially useful in the case of loosely defined domain types for which efficient consensus patterns can not be established. SBASE 2.0 and a set of search and retrieval tools are freely available on request to the authors or by anonymous 'ftp' file transfer from mean value of ftp.icgeb.trieste.it.

The SBASE domain library: a collection of annotated protein segments.

SBASE is a database of annotated protein domain sequences representing various structural, functional, ligand binding and topogenic segments of proteins. The current release of SBASE contains 27,211 entries which are provided with standardized names in order to facilitate retrieval. SBASE is cross-referenced to the major protein and nucleic acid databanks as well as to the PROSITE catalog of protein sequence patterns [Bairoch, A. (1992) Nucleic Acids Res., 20, Suppl., 2013-2118]. SBASE can be used to establish domain homologies through database search using programs such as FASTA [Lipman and Pearson (1985) Science, 227, 1436-1441], FASTDB [Brutlag et al. (1990) Comp. Appl. Biosci., 6, 237-245] or BLAST3 [Altschul and Lipman (1990) Proc. Natl. Acad. Sci. USA, 87, 5509-5513], which is especially useful in the case of loosely defined domain types for which efficient consensus patterns cannot be established. The use of SBASE is illustrated on the DNA binding protein Brain-4. The database and a set of search and retrieval tools are freely available on request to the authors or by anonymous 'ftp' file transfer from < ftp.icgeb.trieste.it >.

Thymopoietins and long postsynaptic neurotoxins share common information in their primary structures.

The informational content of the primary structure of thymopoietin (TP) is investigated using the informational spectrum method (ISM). We show that the sequence of TP shares common information with the sequences of long postsynaptic snake neurotoxins, although no apparent similarity was found among their primary structures. The most sensitive point in the sequence of TP, concerning this information, is D-34, previously determined as being the residue responsible for TP's effect on neuromuscular transmission. Our results suggest that TP and long toxins recognize the neuromuscular nicotinic acetylcholine receptor (AChR) and/or bind to the AChR in a different mode than the short toxins do.