Strain-specific Sequences Research Articles

Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion.

Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like "swarms" of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework.

The variation of dTDP-L-rhamnose pathway genes in Vibrio cholerae.

The genetic variation in the dTDP-L-rhamnose pathway genes (rmlA, rmlB, rmlC and rmlD) in Vibrio cholerae was investigated. The genes are part of the O antigen gene cluster and the aim was to study lateral gene transfer of O antigen gene clusters. The rml genes of an O6 strain were cloned using an Escherichia coli K-12 strain designed for selecting cloned rml genes. Thirty-three strains carrying the known rhamnose-containing O antigens were probed with O6-based rml gene probes, and 19 were positive with from one to all four of the gene probes. Nine rml gene sets from this group were sequenced and found to be in the order rmlBADC, at the 5' end of the gene clusters. A gradient in the level of variation was observed, with highly similar sequences at the 5' end rmlB gene, but very divergent and strain-specific sequences at the 3' end of the rml gene set. The change in level of similarity varied in position, but was always abrupt and coincided with a change in GC content, indicating that the 5' and 3' parts are of different origin, and that recombination within rml genes has occurred. The rml gene sets of two of the strains that did not hybridize with any O6 rml gene probes were also cloned and sequenced. Both gene sets were in the middle of the O antigen gene cluster and were very divergent from each other and all other rml gene sets. This supports the hypothesis that presence of rml genes at the end of the O antigen gene cluster facilitates lateral gene transfer of rml-containing O antigen gene clusters in V. cholerae. The sequence relationships make it possible to identify sites of recombination and to distinguish DNA that has long been in V. cholerae and DNA that probably came into the species with the O antigen gene cluster.

Identification of H. pylori strain specific DNA sequences between two clinical isolates from NUD and gastric ulcer by SSH.

The genomes of Helicobacter pylori (H. pylori) from different individuals are different. This project was to identify the strain specific DNA sequences between two clinical H. pylori isolates by suppression subtractive hybridization (SSH). Two clinical H. pylori isolates, one from gastric ulcer (GU, tester) and the other from non-ulcer dyspepsia (NUD, driver), were cultured and the genomic DNA was prepared and submitted to Alu I digestion. Then two different adaptors were ligated respectively to the 5'-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNA. The un-hybridized tester DNA sequences were amplified by two sequential PCR and cloned into pGEM-T-Easy Vector. The tester strain specific inserts were screened and disease related DNA sequences were identified by dot blotting. Among the 240 colonies randomly chosen, 50 contained the tester strain specific DNA sequences. Twenty three inserts were sequenced and the sizes ranged from 261 bp to 1 036 bp. Fifteen inserts belonged to the H.pylori plasmid pHPO100 that is about 3.5 kb and codes a replication protein A. Other inserts had patches of homologous to the genes of H.pylori in GenBank. Various patterns of dot blots were given and no GU strain unique DNA sequences were found when 4 inserts were used as probes to screen the genomic DNA from 27 clinical isolates, 8 from GU, 12 from duodenum ulcer (DU), 4 from GU-DU, 2 from NUD and 1 from gastric cancer (GC). But a 670 bp DNA fragment (GU198) that was a bit homologous to the 3'-end of the gene of thymidylate kinase was positive in 7 GU strains (7/8), 3 GU-DU strains (3/4) and 3 DU strains (3/12). A 384 bp fragment (GU79) of the replication gene A (repA) was positive only in 4 H.pylori isolates, 2 from GU and 2 from GU-DU. Differences exist in the genes of different H.pylori isolates. SSH is very effective to screen H.pylori strain specific DNA sequences between two clinical isolates, and some of these sequences may have clinical significance.

The Use of Suppression Subtractive Hybridization to Identify Strain-Specific Sequences in Bacterial Genomes

Comparisons of bacterial genomes demonstrate that even strains of one species may strikingly differ in gene set. Strain-specific genes are of considerable interest, as they may be responsible for distinguishing features, such as virulence or drug resistance, of the strain and may be employed as markers in epidemiological or evolutionary studies. Suppression subtractive hybridization (SSH) was shown to be suitable for generating a set of DNA fragments differing between two closely related bacterial strains. More than 95% DNA fragments selected by SSH proved to be specific for Staphylococcus aureus strains ZW compared with strain 29213.

Suppressive subtractive hybridization detects extensive genomic diversity in Thermotoga maritima.

Comparisons between genomes of closely related bacteria often show large variations in gene content, even between strains of the same species. Such studies have focused mainly on pathogens; here, we examined Thermotoga maritima, a free-living hyperthermophilic bacterium, by using suppressive subtractive hybridization. The genome sequence of T. maritima MSB8 is available, and DNA from this strain served as a reference to obtain strain-specific sequences from Thermotoga sp. strain RQ2, a very close relative (approximately 96% identity for orthologous protein-coding genes, 99.7% identity in the small-subunit rRNA sequence). Four hundred twenty-six RQ2 subtractive clones were sequenced. One hundred sixty-six had no DNA match in the MSB8 genome. These differential clones comprise, in sum, 48 kb of RQ2-specific DNA and match 72 genes in the GenBank database. From the number of identical clones, we estimated that RQ2 contains 350 to 400 genes not found in MSB8. Assuming a similar genome size, this corresponds to 20% of the RQ2 genome. A large proportion of the RQ2-specific genes were predicted to be involved in sugar transport and polysaccharide degradation, suggesting that polysaccharides are more important as nutrients for this strain than for MSB8. Several clones encode proteins involved in the production of surface polysaccharides. RQ2 encodes multiple subunits of a V-type ATPase, while MSB8 possesses only an F-type ATPase. Moreover, an RQ2-specific MutS homolog was found among the subtractive clones and appears to belong to a third novel archaeal type MutS lineage. Southern blot analyses showed that some of the RQ2 differential sequences are found in some other members of the order Thermotogales, but the distribution of these variable genes is patchy, suggesting frequent lateral gene transfer within the group.

The cytochrome c maturation locus of Legionella pneumophila promotes iron assimilation and intracellular infection and contains a strain-specific insertion sequence element.

Previously, we obtained a Legionella pneumophila mutant, NU208, that is hypersensitive to iron chelators when grown on standard Legionella media. Here, we demonstrate that NU208 is also impaired for growth in media that simply lack their iron supplement. The mutant was not, however, impaired for the production of legiobactin, the only known L. pneumophila siderophore. Importantly, NU208 was also highly defective for intracellular growth in human U937 cell macrophages and Hartmannella and Acanthamoeba amoebae. The growth defect within macrophages was exacerbated by treatment of the host cells with an iron chelator. Sequence analysis demonstrated that the transposon disruption in NU208 lies within an open reading frame that is highly similar to the cytochrome c maturation gene, ccmC. CcmC is generally recognized for its role in the heme export step of cytochrome biogenesis. Indeed, NU208 lacked cytochrome c. Phenotypic analysis of two additional, independently derived ccmC mutants confirmed that the growth defect in low-iron medium and impaired infectivity were associated with the transposon insertion and not an entirely spontaneous second-site mutation. trans-complementation analysis of NU208 confirmed that L. pneumophila ccmC is required for cytochrome c production, growth under low-iron growth conditions, and at least some forms of intracellular infection. Although ccm genes have recently been implicated in iron assimilation, our data indicate, for the first time, that a ccm gene can be required for bacterial growth in an intracellular niche. Complete sequence analysis of the ccm locus from strain 130b identified the genes ccmA-H. Interestingly, however, we also observed that a 1.8-kb insertion sequence element was positioned between ccmB and ccmC. Southern hybridizations indicated that the open reading frame within this element (ISLp 1) was present in multiple copies in some strains of L. pneumophila but was absent from others. These findings represent the first evidence for a transposable element in Legionella and the first identification of an L. pneumophila strain-specific gene.

In silico discovery of gene-coding variants in murine quantitative trait loci using strain-specific genome sequence databases.

The identification of genes underlying complex traits has been aided by quantitative trait locus (QTL) mapping approaches, which in turn have benefited from advances in mammalian genome research. Most recently, whole-genome draft sequences and assemblies have been generated for mouse strains that have been used for a large fraction of QTL mapping studies. Here we show how such strain-specific mouse genome sequence databases can be used as part of a high-throughput pipeline for the in silico discovery of gene-coding variations within murine QTLs. As a test of this approach we focused on two QTLs on mouse chromosomes 1 and 13 that are involved in physical dependence on alcohol. Interstrain alignment of sequences derived from the relevant mouse strain genome sequence databases for 199 QTL-localized genes spanning 210,020 base-pairs of coding sequence identified 21 genes with different coding sequences for the progenitor strains. Several of these genes, including four that exhibit strong phenotypic links to chronic alcohol withdrawal, are promising candidates to underlie these QTLs. This approach has wide general utility, and should be applicable to any of the several hundred mouse QTLs, encompassing over 60 different complex traits, that have been identified using strains for which relatively complete genome sequences are available.

Epigenetic and Genotype-specific Effects on the Stability ofde Novo Imposed Methylation Patterns in Transgenic Mice

The chloramphenicol acetyltransferase gene under the control of the late E2A promoter of adenovirus type 2 (Ad2) was introduced as transgene into the B6D2F1 mouse strain with mixed genetic background and became extensively de novo methylated. The methylation of this pAd2E2AL-CAT (7-1A) transgene was regulated in a strain-specific manner apparently depending on the site of integration. Transmission of the 7-1A transgene into an inbred DBA/2, 129/sv, or FVB/N genetic background led to a significant loss of methylation in the transgene, whereas C57BL/6, CB20, and Balb/c backgrounds favored the de novo methylation in very specific patterns. The newly established patterns of de novo methylation were transmitted to the offspring and remained stable for many generations, regardless of the heterozygosity of strain-specific DNA sequences present in these mouse strains. Segregation analyses showed a non-mendelian transmission of methylation phenotypes and suggested the involvement of dominant modifiers of methylation. The genotype-specific modifications of the transgene were followed for 11 backcross generations. These observations reflect an evolutionarily conserved mechanism directed against foreign, e.g. viral or bacterial, DNA at least in the chromosomal location of the 7-1A transgene. In seven additional mouse lines carrying the same transgene in different chromosomal locations, strain-specific alterations of methylation patterns were not observed.

Evaluation of strain-specific primer sequences from an abortifacient strain of ovine Chlamydophila abortus ( Chlamydia psittaci) for the detection of EAE by PCR

Strain-specific primer sequences derived from the helicase gene of an ovine abortifacient strain (S26/3) of Chlamydophila abortus ( Chlamydia psittaci) were evaluated for the diagnosis of enzootic abortion in ewes (EAE) by polymerase chain reaction (PCR). C. abortus DNA was amplified from tissues submitted from ovine abortion cases using genus-specific and strain-specific primers in a standard thermal cycler. Amplification was followed by Southern blotting and hybridisation with a strain-specific probe. Real-time PCR was also evaluated using strain-specific primers in a microvolume fluorimeter-based thermal cycler (LightCycler™). Detection using both PCR methods was compared with other diagnostic methods against the standard of McCoy cell culture isolation. In this paper we report the application of strain-specific PCR as a fast, sensitive, specific method for the detection of EAE.

Genetic characterization of the uropathogenic E. coli strain 536--a subtractive hybridization analysis.

ConclusionsThe species E. coli comprises non pathogenic variants and other strains which are able to cause infectious diseases. In order to identify DNA fragments which are present in the pathogenic E. coli strain 536 but absent from the non-pathogenic E. coli strain MG1655 we have performed a subtractive hybridization analysis.We obtained diverse DNA fragments specific for the E. coli strain 536. Sequence data of several cloned subtractive hybridization fragments showed a homology to known virulence genes. Other E. coli strain 536 specific DNA fragments showed no homology to already known DNA or protein sequences and may represent new virulence genes on PAIs.In conclusion subtractive hybridization is a successful method for the identification of strain specific DNA sequences. It is a useful approach to complement other genomic approaches such as proteome analysis or differential RNA display analysis.

Characterization of Satellite DNA Sequences from the Commercially Important Marine Rotifers Brachionus rotundiformis and Brachionus plicatilis.

This study was initiated to search for species-specific and strain-specific satellite DNA sequences for which oligonucleotide primers could be designed to differentiate between various commercially important strains of the marine monogonont rotifers Brachionus rotundiformis and Brachionus plicatilis. Two unrelated, highly reiterated satellite sequences were cloned and characterized. The eight sequenced monomers from B. rotundiformis and six from B. plicatilis had low intrarepeat variability and were similar in their overall lengths, A + T compositions, and high degrees of repeated motif substructure. However, hybridizations to 19 representative strains, sequence characterizations, and GenBank searches indicated that these two satellites are morphotype-specific and population-specific, respectively, and share little homology to each other or to other characterized sequences in the database. Primer pairs designed for the B. rotundiformis satellite confirmed hybridization specificities on polymerase chain reaction and could serve as a useful molecular diagnostic tool to identify strains belonging to the SS morphotype, which are gaining widespread usage as first feeds for marine fish in commercial production.

Pollen-related food allergy: cloning and immunological analysis of isoforms and mutants of Mal d 1, the major apple allergen, and Bet v 1, the major birch pollen allergen.

Mal d 1, the major apple allergen, cross-reacts with IgE specific for the major birch pollen allergen, Bet v 1, and is responsible for birch pollen related food allergy to apple. Isoforms of Bet v 1 showing minor sequence variations display different binding capacity for specific IgE antibodies from allergic patients. Moreover, strain-dependent variation of allergenicity has been reported for apples. To investigate the occurrence of strain-dependent isoforms of Mal d 1 which may differ in their allergenic potential, to obtain data on structures essential for binding of Mal d 1 to the antibody, and to gain insights into the structures responsible for its IgE cross-reactivity to Bet v 1. The cDNA of Mal d 1 from various apple strains was amplified by a PCR strategy based on conserved regions of known Mal d 1-sequences, and sequenced. Two major isoforms of Mal d 1 were expressed as recombinant proteins and purified, as were different variants of the major birch pollen allergen, Bet v 1. Together with already existing recombinant birch pollen and apple allergens, these were subjected to allergenicity testing by IgE-immunoblotting, enzyme allergo sorbent test and dose related mediator release. "Hot-spots" for IgE-reactivity were identified by site-directed mutagenesis. Twelve Mal d 1-clones were sequenced from 7 apple varieties and compared to 3 known Mal d 1 sequences. The clones were clustered into two groups, each showing a high degree of sequence identity to one of the known sequences and specific differences to the third sequence. No strain-specific sequences were identified. In contrast, apple strains with reported differences in allergenicity showed different expression levels of the major allergen. Immunologic testing of recombinant allergens revealed high IgE binding capacity of 2 major isoforms, named GD26 and GS29, with a slightly higher IgE binding capacity of GD26. Moreover, the allergenicity was similar to another r Mal d 1 reported in the literature, representing the isoform divergent from our clones. Mutational analysis of our Mal d 1 allergens identified serine in position 111 as essential for IgE binding. Allergenicity was almost depleted by changing this residue into a proline. Moreover, the corresponding serine residue, present in position 112 of Bet v 1, was in a similar manner crucial for the allergenicity of the birch pollen allergen. We conclude that divergent allergenicity of apple strains mainly depends on different expression levels of the major allergen. Introduction of a proline residue in position 111 of Mal d 1 and in position 112 of Bet v 1 led to a drastic reduction of allergenicity of both the pollen and the food allergen, obviously also removing the cross-reactive epitope. Mutants with reduced IgE-reactivity but maintained T-cell reactivity may represent new candidates for a safer specific immunotherapy with reduced side-effects.

Characterisation of strain-specific sequences from an abortifacient strain of ovine Chlamydia psittaci using subtraction hybridisation

Enzootic abortion in ewes (EAE) is caused by strains of Chlamydia psittaci which have the ability to invade and colonise the placenta of sheep. In an attempt to improve diagnostic methods for the detection of EAE, subtraction hybridisation was used to isolate unique fragments of the genome of an abortifacient strain (S26/3) of C. psittaci. One S26/3 strain-specific sequence identified was shown to encode a putative helicase which is repeated throughout the EAE genome. The labelled strain-specific helicase gene fragment was used in a dot-blot hybridisation test for the detection of EAE DNA in ovine placental samples. We report the identification of C. psittaci S26/3 strain-specific sequences with potential as diagnostic probes for the detection of EAE.

Immune Response to an Epitope of the NS4 Protein of Hepatitis C Virus in HCV-Related Disorders

NS4, a nonstructural protein of HCV, is a frequent target of antibodies in infected subjects. According to recent data, the antibodies frequently recognize the sequence 1921–40 of the NS4 protein. The aim of this work was to analyze antibody reactivity with the sequence 1921–40 in different HCV-related disorders. Although this sequence is located in a relatively invariant region of viral genome, two strain-specific sequences are described. Thus, three NS4 1921–1940 peptides were synthesized: the BK shared by most viral strains, the J6 (strain 2a), and the J8 (strain 2b). The peptides were used as antigens in the solid phase for measuring serum IgG antibodies in an ELISA assay. Antibodies reactive with the 1921–40 BK peptide were detected in 64% of sera from patients with autoimmune hepatitis (AIH), 51% from chronic hepatitis C (CHC), and 22% from mixed cryoglobulinemia (MC). The frequency of positive sera in MC was significatively lower than in AIH (P< 0.0001) or CHC (P< 0.0021). Similar results were obtained with the J6 and J8 peptides. All sera that did not react with the BK peptide were negative on J6 and J8 and conversely most sera reacting with the BK peptide also bound the J6 and the J8 peptides. No correlation was found between the genotype of the infecting virus and the presence of antibodies to any of the NS4 peptides. These results indicate that many HCV-infected subjects produce antibodies to the NS4 sequence 1921–40. The immune response to this sequence is not strain specific and varies with the different disorders associated with HCV infection.

Differential genome analysis of bacteria by genomic subtractive hybridization and pulsed field gel electrophoresis.

A comprehensive analysis of the differences between the genomes of two closely related bacterial strains should give insight into the molecular basis of their individual phenotypic and genotypic characteristics. Here we present an integrative approach including two different strategies for the thorough investigation of genomic divergence. We have combined two techniques including genomic subtractive hybridization and comparative genome mapping by pulsed field gel electrophoresis (PFGE) techniques. The subtractive method for which a protocol is given herein results in the production of a library of specific DNA sequence tags present only in one strain, while the construction of macrorestriction maps of the bacterial chromosomes yields data about the overall genome organization and the arrangement and distance of gene loci. Comparison of the physical and genetic maps and determination of the map positions of the strain-specific DNA sequences reveals gross chromosomal modifications, insertions or deletions of additional genetic material, and transpositional events. The further investigation of the strain-specific regions yields information about the nature and origin of the acquired DNA and their influence on the evolution of the individual bacterial genome. The two methods were applied to differential genome analysis of clonal divergence in Pseudomonas aeruginosa choosing two clone C isolates from diverse habitats.

Chromosomal regions specific to pathogenic isolates of Escherichia coli have a phylogenetically clustered distribution.

We studied the ancestry of virulence-associated genes in Escherichia coli by examining chromosomal regions specific to pathogenic isolates. The four virulence determinants examined were the alpha-hemolysin (hly) loci hlyI and hlyII, the type II capsule gene cluster kps, and the P (pap) and S (sfa) fimbria gene clusters. All four loci were shown previously to be associated with pathogenicity islands of uropathogenic E. coli isolates. The hly, kps, sfa, and pap regions each have an unexpected clustered distribution among the E. coli collection of reference (ECOR) strains, but all these regions were absent from a collection of diarrheagenic E. coli isolates. Strains in the ECOR subgroup B2 typically had a combination of at least three of the four loci, and all strains in subgroup D had a copy of the kps and pap clusters. In contrast, only four strains in subgroup A had either hly, kps, sfa, or pap, and no subgroup A strains had all four together. Strains of subgroup B1 were devoid of all four virulence regions, with the exception of one isolate that had a copy of the sfa gene cluster. This phylogenetic distribution of strain-specific sequences corresponds to the ECOR groups with the largest genome size, namely, B2 and D. We propose that the pathogenicity islands are ancestral to subgroups B2 and D and were acquired after speciation, with subsequent horizontal transfer into some group A, B1, and E lineages. These results suggest that the hly, kps, sfa, and pap pathogenicity determinants may play a role in the evolution of enteric bacteria quite apart from, and perhaps with precedence over, their ability to cause disease.

Molecular characterization of a strain-specific sequence in Campylobacter jejuni.

Campylobacter jejuni strains 81116 and NCTC 11392 were shown to contain a region of DNA that was not present in other strains. This region was cloned from the chromosome of strain 81116 and the nucleotide sequence determined. It was found to contain an insert of 742 bp which was flanked by direct repeats of 45 bp. Although the nature of this sequence is unknown at present, it is clear that it only presents in certain strains of Camp. jejuni and it may therefore be useful as an epidemiological tool.

Identification of strain-specific nucleotide sequences in E1 and NS4 genes of rubella virus vaccine strains in Japan

Strain-specific nucleotide sequences of E1 and NS4 genes in five strains of a live rubella virus vaccine manufactured in Japan were identified for comparison, using 2389 nucleotides (1443 nucleotides of the E1 gene, 41 of the 3′ terminal region following the E1 gene and 905 of the NS4 gene). Sequences of the E1 gene in three strains (Matsuura, TCRB19 and To-336) were identified. Takahashi and Matsuba strains shared common sequences, but were discriminated by the sequence of the NS4 gene. These five strains showed a phylogenetic relationship with the places of their isolation. In a comparative study of three strains with their unattenuated progenitors, the nucleotides in these regions were almost conserved during the attenuation process.

Constitutive Expression of Murine Complement Factor B Gene Is Regulated by the Interaction of Its Upstream Promoter with Hepatocyte Nuclear Factor 4

Factor B (Bf) is a constituent of the alternative pathway of complement activation encoded within the major histocompatibility complex. Transcription of the murine gene from two initiation sites generates two Bf mRNA species differing in size and tissue distribution. Striking genetic, tissue-specific differences in Bf mRNA levels at extrahepatic sites (kidney and intestine) among mouse strains correlate with a DNA sequence polymorphism in the 5'-flanking region of the gene and differential nuclear protein binding at the Bf upstream transcriptional initiation site (UIS). To ascertain the functional consequences of this polymorphism in the Bf promoter, we analyzed the effects of strain-specific sequences in the Bf 5' region on the expression of a chloramphenicol acetyltransferase (CAT) reporter gene transfected in human and mouse hepatoma cells. The CAT activity and mRNA level produced when transcription was driven by the sequence of strains with high extrahepatic expression were reduced to background levels when the sequence specific to the low expressor strains was used. Eighty percent of this difference was accounted for by a point substitution that affects DNA-protein interaction at the UIS, the sequence of higher affinity conferring higher expression. Hepatocyte nuclear factor 4 (HNF-4), derived from HepG2, mouse liver and kidney or cell-free translation of HNF-4 RNA, is the nuclear protein that preferentially binds to the high expressor UIS. Bf-CAT is not expressed in cells that lack HNF-4 (CV-1). However, co-transfection of HNF-4 into CV-1 cells drives Bf-CAT expression and reproduces the differences derived from the substitution that affect HNF-4 binding in vitro. These data show that interaction of HNF-4 with polymorphic variants of the upstream Bf promoter is the major determinant of strain-specific extrahepatic factor B expression.

Biological Testing versus Sequence Analysis for Determination of Drug Resistance of Human Immunodeficiency Virus

Resistance of HIV-1 to 3'-azido-3'-deoxythymidine (AZT) is associated with one or more of five mutations in the reverse transcriptase (RT) segment of the polymerase gene ("AZT-specific mutations"). Therefore, sequence analysis of the proviral DNA, derived directly from the blood, is considered to replace the biological test. Additional arguments are non-cultivatable viral strains, the universality of the sequence analysis in combination therapy, and the suspicion that the cultivated virus does not represent the predominant viral variant in the blood. In this investigation, 21 strains of human immunodeficiency virus type 1 (HIV-1) were comparatively analysed by molecular and biological testing. For 12 strains, the homology of the RT gene segment between the predominant provirus in the blood and the cultivated virus was ascertained (99.72% homology). 11 strains from untreated patients or patients treated no longer than 5 months were free from AZT-specific mutations and proved to be sensitive. 10 strains from patients treated for 17 to 57 months displayed 2-4 AZT-specific mutations. However, it was not possible to correlate the degree of sensitivity to the number or the pattern of the mutations. Suppression of AZT resistance by strain-specific sequences in other parts of the gene are discussed as the reason for that discrepancy. Remarkably, the productivity of resistant virus strains could be drastically enhanced by non-inhibiting concentrations of the drug.