Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.     

Determination of genetic diversity among Indian isolates of Rhizoctonia bataticola causing dry root rot of chickpea

Genetic diversity among 27 isolates (23 from chickpea and 4 from other host crops) of Rhizoctonia bataticola representing 11 different states of India was determined by random amplified polymorphic DNA (RAPD), internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) and ITS sequencing. The isolates showed variability in virulence test. Unweighted paired group method with arithmetic average cluster analysis was used to group the isolates into distinct clusters. The clusters generated by RAPD grouped all the isolates into six categories at 40% genetic similarity. High level of diversity was observed among the isolates of different as well as same state. Some of the RAPD (OPN 4, OPN 12, and OPN 20) markers clearly distinguished majority of the isolates into the area specific groups. The ITS I, 5.8rDNA and ITS II regions of 11 isolates representing different RAPD groups were amplified with primers ITS 1 and ITS 4 and digested with seven restriction enzymes. The restriction enzymes DraI, MboI, RsaI, and AluI were found to be suitable for differentiating the isolates into five categories by showing isolate specific ITS-RFLP patterns. The isolates were variable in their nucleotide sequences of the ITS regions. This is the first study on genetic diversity among chickpea isolates of R. bataticola.     

Identification of enterohepatic Helicobacter species by restriction fragment-length polymorphism analysis of the 16S rRNA gene

Background. Restriction fragment-length polymorphism (RFLP) analysis of a 1,200-bp polymerase chain reaction–amplified DNA fragment of gene coding for 16S rRNA was used to generate restriction profiles of 11 enterohepatic Helicobacter spp. isolated from various animals and humans.
Methods. The amplicon from each Helicobacter sp. was digested with four restriction endonucleases: Alu I, Hinf I, Hha I, and Dde I. Alu I digestion produced five patterns that were useful for initial differentiation.
Results. Most Helicobacter spp. isolated from rodents had the same RFLP profiles by Alu I digestion (except H. rodentium and H. cholecystus ), but they had different RFLP profiles by Hha I digestion. Only H. bilis and " H. rappini" mouse isolates could not be readily distinguished by the polymerase chain reaction-RFLP method. However, these two species can be distinguished using H. bilis specific primers. Some of the Helicobacter spp. have an intervening sequence in their 16S rRNA gene, which changes the RFLP patterns; in these cases, sequencing is the preferred method to make an appropriate diagnosis.
Conclusions. The RFLP method used in this study was straightforward and rapid and should prove useful as an adjunct for identification and classification of multiple enterohepatic Helicobacter spp.     

Identification of blood meal sources of Lutzomyia longipalpis?using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.     

Polymerase chain reaction amplification and restriction fragment length polymorphism analysis of 16S rRNA genes from methanogens

For restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes, the rDNA fragments of 1.5 kb were amplified by polymerase chain reaction (PCR) from crude cell lysates of various methanogenic species which were prepared by a combined technique of ultrasonic treatment and protease digestion. The PCR products were purified by the polyethylene glycol precipitation method and treated with various restriction enzymes. The 16S rDNA fragments digested with HaeIII or HhaI gave species-specific RFLP profiles on simplified agarose gel electrophoresis. 16S rDNA gragments of 0.4 kb from the bulk DNA extracted from mixed populations of anaerobic sludge were also amplified by PCR with a pair of methanogen-specific primers and cloned directly by the T-A cloning technique. The cloned 16S rDNAs from recombinants were reamplified by PCR, and RFLP pattern analysis was performed following digestion with HhaI. The PCR-RFLP analysis of 16S rDNA with the present protocol can be completed within one day, provided that sufficient amounts of test cells are available, and has great promise as a simple and rapid technique for identification of methanogens. A combined method consisting of PCR amplification, direc cloning with T vectors, and RFLP analysis of 16S rDNA is also useful for rapid estimation of the mixed population structure of methanogens without the need for cultivation and isolation.     

Restriction site polymorphisms in the pig β-globin gene cluster

A restriction fragment length polymorphism was detected in pig DNA digested with Hind III restriction endo nuclease and probed with rabbit β₁-globin gene. Eight different phenotypes were observed and for six of them family data demonstrated that they are determined by three alleles. As this polymorphism is not found with four other restriction endo nucleases (Bam HI, Eco RI, Kpn I, and Pst I), single point mutations are proposed to explain the observed differences.     

Restriction site polymorphisms in the pig beta-globin gene cluster

A restriction fragment length polymorphism was detected in pig DNA digested with Hind III restriction endonuclease and probed with rabbit beta 1-globin gene. Eight different phenotypes were observed and for six of them family data demonstrated that they are determined by three alleles. As this polymorphism is not found with four other restriction endonucleases (Bam HI, Eco RI, Kpn I, and Pst I), single point mutations are proposed to explain the observed differences.     

Detection and Identification of cry1I Genes in Bacillus thuringiensis Using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Analysis

A polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method for detection and identification of cry1I genes from Bacillus thuringiensis (Bt) was established. Based on the analysis of conserved regions of the cry1I genes, 2 oligonucleotide primers were designed to amplify a 665-bp fragment of the genes. The amplification products were digested with restriction endonuclease HinfI or with RsaI in addition for specific detection of different variants from the known subclasses of cry1I genes. The PCR-RFLP pattern obtained revealed the detection of cry1I genes in 151 of 202 native Bt isolates. Furthermore, cry1I genes were detectable in 10 of 19 standard strains tested. The cry1Ia gene was the most abundant cry1I gene subclass present in 54 of 56 native Bt isolates and in 8 of 10 standard strains. Based on the results obtained, the PCR-RFLP method may be a valuable and reliable tool for specific detection and identification of cry1I genes.     

Echinococcus granulosus genotypes in livestock of Iran indicating high frequency of G1 genotype in camels

In this study, 112 Echinococcus granulosus isolates from different livestock of Iran were genotyped by PCR amplification of ribosomal DNA-internal transcribed spacer 1 (rDNA-ITS1) region followed by restriction fragment length polymorphism (RFLP) with the enzyme RsaI. The possibility of intra-genotype variation was also investigated using randomly amplified polymorphic DNA (RAPD) analysis. Isolates from sheep, goats, cattle and the majority of camels (12 of 18; 66.7%) were identified as the G1 genotype and a few camel isolates (6 of 18; 33.3%) belonged to the G6 genotype. Overall G1 and G6 genotypes were identified in 94.6% (106 of 112) and 5.3% (6 of 112) of all isolates, respectively. RAPD analysis based on 15 separate primers showed 7-14 bands of 200-3000 bp for strain G1. Considering each individual primer, no differences observed among isolates from different hosts and between livers and lungs. This study confirmed the existence of G1 and G6 genotypes in Iran. Moreover, G1 is much more prevalent even in camels, indicating the importance of sheep-dog cycle in public health. Studying intra-genotypic variation of E. granulosus warrants more research using other primers and methods.     

Restriction fragment length polymorphism of the major histocompatibility complex of the dog

Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D-homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (Bam HI, Eco RI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I, and Bgl II), separated by agarose gel electrophoresis, and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabeled HLA cDNA probes corresponding to DR, DQ, DP, and DO beta genes. The autoradiograms for all nine enzyme digests displayed multiple bands with the DRb, DQb, and DPb probes while the DOb probe hybridized with one to two bands. The RFLP patterns were highly polymorphic but consistent within each DLA-D type. Standard RFLP patterns were established for nine DLA-D types which could be discriminated from each other by using two enzymes (Rsa I and Pst I) and the HLA-DPb probe. Cluster analysis of the polymorphic restriction fragments detected by the DRb probe revealed four closely related supertypic groups or DLA-DR families: Dw3+Dw4+D1, Dw8+D10, D7+D16+D9, and Dw1. This study provides the basis for DLA-D genotyping at a population level by RFLP analysis. These results also suggest that the genetic organization of the DLA-D region may closely resemble that of the HLA complex.     

A contribution to the subgeneric systematics of Oligoryzomys (Rodentia,Muridae) from Argentina by means of PCR–RFLP patterns of mitochondrial DNA

Subgeneric recognition of rodents of the genus Oligoryzomys from Argentina is specially difficult because morphologic and morphometric criteria to identify species are poorly defined. In order to contribute to the unequivocal identification and geographic distribution of Oligoryzomys species, we studied the restriction fragment length polymorphism (PCR-RFLP) patterns of the D-loop region of the mitochondrial DNA in specimens collected from different regions of Argentina. PCR amplification products of 59 individuals were digested with five restriction enzymes. Patterns obtained were used to produce two phylogenetic consensus trees, one obtained with PAUP and the other with MIX analyses. All specimens from the Patagonia (southern region) clustered together and would correspond to O. longicaudatus. Individuals from the North grouped in four clusters (two of them comprising one individual), supported by high bootstrap values. The one including the 82% of the rodents from the northern region would correspond to the species O. chacoensis.     

Characterization of Gibberella fujikuroi complex isolates by fumonisin B1 and B2 analysis and by RAPD and restriction analysis of PCR-amplified internal transcribed spacers of ribosomal DNA

Twenty nine isolates of Fusarium spp. (twenty four of them belonging to the Gibberella fujikuroi complex) isolated from banana and corn from different geographical regions were analyzed for their ability to produce fumonisins B1 and B2 and for genetic relatedness using random amplified polymorphic DNA (RAPD) and restriction analysis of PCR amplification products of the 5.8s ribosomal DNA-intervening internal transcribed spacer regions (ITS I-5.8S-ITS II). For RAPD analysis, six of twenty oligonucleotide primers were selected after testing with five Fusarium spp. isolates and used to characterize 24 additional isolates. DNA fragments from the 29 isolates of Fusarium spp., which were approximately 560 bp, were amplified with the universal primers ITS1 and ITS4. The restriction enzymes HaeIII, MboI, HpaII and MspI were useful for distinguishing the isolates. The RAPD analysis permitted to find interspecific differences among the isolates of Fusarium spp., between isolates with low and high capacity of fumonisin production and among isolates from different hosts. The restriction fragment length polymorphism (RFLP-PCR) analysis permitted to distinguish among different species of Fusarium. In combination with morphological analysis, the results of this research may find an application for the diagnosis of unknown Fusarium spp. and, particularly, for the characterization of fumonisin-producing isolates, which may be very useful in the food technology field.     

Population Variability of the FimH Type 1 Fimbrial Adhesin in Klebsiella pneumoniae

FimH is an adhesive subunit of type 1 fimbriae expressed by different enterobacterial species. The enteric bacterium Klebsiella pneumoniae is an environmental organism that is also a frequent cause of sepsis, urinary tract infection (UTI), and liver abscess. Type 1 fimbriae have been shown to be critical for the ability of K. pneumoniae to cause UTI in a murine model. We show here that the K. pneumoniae fimH gene is found in 90% of strains from various environmental and clinical sources. The fimH alleles exhibit relatively low nucleotide and structural diversity but are prone to frequent horizontal-transfer events between different bacterial clones. Addition of the fimH locus to multiple-locus sequence typing significantly improved the resolution of the clonal structure of pathogenic strains, including the K1 encapsulated liver isolates. In addition, the K. pneumoniae FimH protein is targeted by adaptive point mutations, though not to the same extent as FimH from uropathogenic Escherichia coli or TonB from the same K. pneumoniae strains. Such adaptive mutations include a single amino acid deletion from the signal peptide that might affect the length of the fimbrial rod by affecting FimH translocation into the periplasm. Another FimH mutation (S62A) occurred in the course of endemic circulation of a nosocomial uropathogenic clone of K. pneumoniae. This mutation is identical to one found in a highly virulent uropathogenic strain of E. coli, suggesting that the FimH mutations are pathoadaptive in nature. Considering the abundance of type 1 fimbriae in Enterobacteriaceae, our present finding that fimH genes are subject to adaptive microevolution substantiates the importance of type 1 fimbria-mediated adhesion in K. pneumoniae.Klebsiella pneumoniae is recognized as an important opportunistic pathogen that frequently causes urinary tract infections (UTI), septicemia, or pneumonia, particularly in immunocompromised individuals (25). K. pneumoniae is responsible for up to 10% of all nosocomial bacterial infections (12, 35). In recent years, a high incidence of community-acquired K. pneumoniae pyogenic liver abscess with a high mortality rate has been reported, especially from Taiwan, but also from other Asian countries, Europe, and North America (6, 8, 19, 27, 44). Furthermore, 15% to 30% of K. pneumoniae isolates are resistant to broad-spectrum cephalosporins via plasmid-encoded extended-spectrum β-lactamases (5).In contrast to many other bacterial pathogens, K. pneumoniae is ubiquitous in nature. Its nonclinical habitats include environmental locations, such as vegetation, soil, and surface waters, as well as transient commensal colonization of mucosal surfaces in humans and other animals (1). Several studies have reported K. pneumoniae isolates of environmental origin to be nearly identical to clinical isolates with respect to several phenotypic properties (16, 22, 23, 25, 30). It has been suggested that environmental isolates of K. pneumoniae may be as virulent as clinical isolates (24, 39).Several virulence factors have been identified in K. pneumoniae (25, 38). The prominent polysaccharide capsule expressed by most isolates, together with the lipopolysaccharide layer, protects the bacteria against phagocytosis and the bactericidal activity of serum. Fimbrial adhesins expressed by the bacteria are protein structures able to recognize molecular receptors and to facilitate adherence to specific tissue surfaces in the host. K. pneumoniae produces two major fimbrial adhesion organelles, type 1 and type 3 fimbriae (9). Type 1 fimbriae have mannose-sensitive hemagglutinins, while type 3 fimbriae have mannose-resistant hemagglutinins (21).Type 1 fimbriae are the most common adhesive organelle in Enterobacteriaceae and have been most extensively studied in Escherichia coli. The type 1 fimbrial structures of K. pneumoniae are homologous to those of E. coli with regard to genetic composition and regulation (37). Type 1 fimbriae and the adhesive subunit FimH, in particular, play an important role in UTI caused by both K. pneumoniae and E. coli (3, 15, 17, 30, 37). Analysis of E. coli fimH variation at the population level has revealed that the FimH adhesin in urinary E. coli isolates accumulates amino acid replacements that increase its tropism toward the uroepithelium and various components of basement membranes (14, 26, 31, 33, 46). Most of the replacements increase the monomannose binding capability of FimH under low shear by altering allosteric catch bond properties of the protein (40). The natural FimH mutants were shown to provide an advantage in colonization of the urinary tract in a mouse model (32) and correlate with the overall extraintestinal virulence of E. coli (11). Thus, FimH mutations are pathoadaptive in nature. No such population-wide analysis has been performed for K. pneumoniae fimH.Population genetic analysis involves comparison of the nucleotide and structural variability of the locus of interest across multiple bacterial strains of different clonalities and geographic origins. The clonal structure of the strains can be determined by multiple-locus sequence typing (MLST), in which 400- to 500-bp sequences of multiple genetically unlinked loci are determined in order to define the phylogenetic relationship of the strains and the extent of interclonal gene recombination (horizontal gene transfer). MLST has been used to reveal the epidemiological relationship of ceftazidime- and ciprofloxacin-resistant K. pneumoniae isolates of nosocomial origin (4). In addition, the analysis of gene variability enables the determination of the type of selection processes acting on loci of interest, with possible identification of mutational changes of functional significance that could enhance the organism''s ability to cause disease, i.e., that could be of a pathoadaptive nature.In this study, the population dynamics of the K. pneumoniae FimH adhesin were determined by analysis of fimH allelic diversity in strains of environmental and various clinical origins in the context of K. pneumoniae clonal structure based on the allelic diversity of three loci—tonB, mdh and fumC—commonly used for MLST.     

Molecular characterization of Pisolithus spp. isolates by rDNA PCR-RFLP

 Variation within ribosomal DNA (rDNA) genes of 19 isolates of Pisolithus from different geographic origins and hosts was examined by polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism (RFLP) analysis. The primers utilized amplify rDNA regions in a wide range of fungi. One amplified region includes the internal transcribed spacer (ITS), which has a low degree of conservation. The ITS amplification products (640–750 bp) were digested with a variety of restriction endonucleases. Cluster analysis based on the restriction fragments grouped the isolates into three distinct groups: group I contained isolates collected in the northern hemisphere, except Pt 1, group II contained those collected in Brazil and group III contained isolate Pt 1. Additional analysis of other rDNA regions, IGS, 17 S and 25 S rDNA, resulted in similar groups. The data suggest that the taxonomy and systematics of this ectomycorrhizal fungus should be revised. Accepted: 16 September 1998     

Survey of Paxillus involutus (Batsch) Fr. inoculum and fruitbodies in a nursery by IGS-RFLPs and IGS sequences

Maintenance and stress resistance of beech and oak plantlets is considerably improved when seedlings are inoculated in the nursery with Paxillus involutus, but success of mycorrhization is strongly related to the fungal isolate. To determine the success of individual P. involutus isolates used for inoculation, a molecular characterization was carried out and used to survey the mycorrhizas and the fruitbodies obtained in the nursery. Persistency of isolates was also surveyed on mycorrhizas 1 and 2 years after planting the seedlings to the field. Molecular characters for practical survey of the isolates were the length of the PCR product using primers CLN12/5SA for amplification and the restriction fragment length polymorphism of the intergenic spacer (IGS1) digested by HaeIII. Sequencing of the IGS1 confirmed the length polymorphisms, but did not allow further discrimination of the isolates. Differences in IGS1-sequences between isolates increased with larger geographical distance of isolate origin, but genetically closely related isolates could not be separated. The molecular survey indicated that the P. involutus inoculants were not necessarily those on the mycorrhizas, but some were more competitive than others. Only the most efficient isolate was found to form mycorrhizas with a plant and within a nursery plot when several isolates were mixed for inoculation. An Australian P. involutus isolate (ANU) failed in mycorrhization of beech and oak in the nursery and the seedlings became mycorrhizal by another isolate. Fruitbody production in the nursery could be linked to the inoculated isolates. The isolates remained associated with the inoculated beech and oak for 2 years after outplanting to a forest area and arable land. The molecular survey, thus, helped to select efficient isolates of P. involutus for forestry application. Received: 15 July 1999 / Accepted: 6 December 1999     

中国东部栗疫病菌群体的遗传分化*

本实验采用RFLP技术,对中国东部栗疫病菌(Cryphonectria parasitica)进行了群体遗传结构的研究。313个参试菌株来自10个省(市)的16个群体(子群体),样本分布在北纬24°N—41°N。各菌株的DNA分别用限制性内切酶Pst Ⅰ和EcoR Ⅰ酶切,先后以10个低拷贝DNA探针和1个DNA指纹图谱探针进行了杂交和检测。结果表明,两个探针(pCB29和pMS29.1)的杂交图谱呈单态性;探针pCB19的杂交图谱显示,菌株DNA以PstⅠ酶切的为单态性,以EcoR Ⅰ酶切的则呈多态性;其他7个低拷贝探针的杂交图谱都呈多态性(Pst Ⅰ酶切)、指纹图谱探针的检测结果显示,辽宁凤城群体的菌株与中国东部其他群体的菌株相比,具有更多的限制性杂交片段,菌株间的遗传变异性也更大。     

ITS-RFLP fingerprinting and molecular marker for detection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">ciceris</Emphasis>

Genetic diversity of 11 representative isolates of Fusarium oxysporum f.sp. ciceris causing chickpea wilt was determined through internal transcribed spacer (ITS) region of the ribosomal DNA-restriction fragment length polymorphism (ITS-RFLP). ITS1+5.8s+ITS2 regions of the isolates were amplified with a set of primers ITS1 and ITS4 and amplified products were digested with 4 restriction enzymes (AluI, MboI, RsaI, MseI). Six different kinds of ITS-RFLP patterns were obtained. The ITS region of these isolates was sequenced and deposited to NCBI GeneBank. The nucleotide sequence homology of ITS region grouped the isolates into 5 categories. Primers were designed with sequence information using Primer 3 software. F. oxysporum f.sp. ciceris specific markers (FOC F2 and FOC R2) based on ITS region were developed for the first time for detection of the pathogen. The markers produced an amplicon of 292 bp; they were validated against the isolates of the pathogen collected from different locations of India.     

Large-Restriction-Fragment Polymorphism Analysis of Mycobacterium chelonae and Mycobacterium terrae Isolates

Mycobacterium chelonae and Mycobacterium terrae were reported to be frequently present in the environment of the Mycobacterium bovis BCG trial area in south India. Six isolates of M. chelonae and four isolates of M. terrae obtained from different sources in this area were analyzed by pulsed-field gel electrophoresis (PFGE) to examine large-restriction-fragment (LRF) polymorphism using the chromosomal DNA digested with DraI and XbaI restriction enzymes. With the exception of one isolate of M. terrae, DNA from all other isolates could be digested with DraI and XbaI and resulted in separable fragments. Visual comparison of the LRFs showed a unique pattern for each of the isolates tested. A computer-assisted dendrogram of the percent similarity demonstrated a high degree of genetic diversity in this group of isolates. This study demonstrates that species of nontuberculous mycobacteria, particularly M. chelonae and M. terrae, can be successfully typed by their LRF pattern using PFGE, which does not require species-specific DNA probes.     

A new methodology combining PCR, cloning, and sequencing of clones discriminated by RFLP for the study of microbial populations: application to an UASB reactor sample

This work describes a methodology combining DNA extraction, polymerase chain reaction amplification with primers targeting 16S ribosomal RNA genes, cloning, and sequencing of clones previously analyzed by restriction fragment length polymorphism (RFLP), which can be applied to study the microbial diversity in a given habitat. The methodology allows the minimization of the sequencing effort, which is particularly relevant when analyzing large numbers of clones. The methodology does not require particularly skilled personnel and can easily be adaptable to the molecular characterization of virtually any particular microbial population, provided that both adequate primers and suitable restriction enzymes for RFLP analysis of the clone library have been chosen. An example of application is presented, in which a sample taken from a continuously operating upflow anaerobic sludge blanket reactor was analyzed. RFLP analysis of the initial 162 clones with HaeIII allowed the identification of only 28 distinct profiles. As expected, identical RFLP profiles corresponded to identical nucleotide sequences.