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Osteogenesis imperfecta (OI), is a genetic disorder of bone fragility caused by mutations in collagen I or proteins involved in collagen processing. Previous studies in mice and human OI bones have shown that excessive activation of TGF-β signaling plays an important role in dominant and recessive OI disease progression. Inhibition of TGF-β signaling with a murine pan-specific TGF-β neutralizing antibody (1D11) was shown to significantly increase trabecular bone volume and long bone strength in mouse models of OI. To investigate the frequency of dosing and dose options of TGF-β neutralizing antibody therapy, we assessed the effect of 1D11 on disease progression in a dominant OI mouse model ( col1a2 gene mutation at G610C). In comparison with OI mice treated with a control antibody, we attempted to define mechanistic effects of 1D11 measured via μCT, biomechanical, dynamic histomorphometry, and serum biomarkers of bone turnover. In addition, osteoblast and osteoclast numbers in histological bone sections were assessed to better understand the mechanism of action of the 1D11 antibody in OI. Here we show that 1D11 treatment resulted in both dose and frequency dependency, increases in trabecular bone volume fraction and ultimate force in lumbar bone, and ultimate force, bending strength, yield force, and yield strength in the femur ( p  ≤ 0.05). Suppression of serum biomarkers of osteoblast differentiation, osteocalcin, resorption, CTx-1, and bone formation were observed after 1D11 treatment of OI mice. Immunohistochemical analysis showed dose and frequency dependent decreases in runt-related transcription factor, and increase in alkaline phosphatase in lumbar bone sections. In addition, a significant decrease in TRACP and the number of osteoclasts to bone surface area was observed with 1D11 treatment. Our results show that inhibition of the TGF-β pathway corrects the high-turnover aspects of bone disease and improves biomechanical properties of OI mice. These results highlight the potential for a novel treatment for osteogenesis imperfecta. © 2021 Sanofi-Genzyme. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research., Competing Interests: The authors all declare there is no conflict of interest regarding the publication of this article. All authors, unless stated otherwise, are employees of Sanofi. Oxana Beskrovnaya and Ryan Russo are currently employed with Dyne Therapeutics, Shannon Dwyer with Artax Biopharma, Nikolai Bukanov with Janssen Pharmaceuticals, Jeffry Nyman and Sasidhar Uppuganti with Vanderbilt University, and Yves Sabbagh with Inozyme Pharma. Yves Sabbagh is also a shareholder of Sanofi., (© 2021 Sanofi‐Genzyme. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.)

Following the discovery of (R)-roscovitine's beneficial effects in three polycystic kidney disease (PKD) mouse models, cyclin-dependent kinases (CDKs) inhibitors have been investigated as potential treatments. We have used various affinity chromatography approaches to identify the molecular targets of roscovitine and its more potent analog (S)-CR8 in human and murine polycystic kidneys. These methods revealed casein kinases 1 (CK1) as additional targets of the two drugs. CK1ε expression at the mRNA and protein levels is enhanced in polycystic kidneys of 11 different PKD mouse models as well as in human polycystic kidneys. A shift in the pattern of CK1α isoforms is observed in all PKD mouse models. Furthermore, the catalytic activities of both CK1ε and CK1α are increased in mouse polycystic kidneys. Inhibition of CK1ε and CK1α may thus contribute to the long-lasting attenuating effects of roscovitine and (S)-CR8 on cyst development. CDKs and CK1s may constitute a dual therapeutic target to develop kinase inhibitory PKD drug candidates.

Sevelamer hydrochloride, a noncalcium phosphate binder, has been shown to reduce coronary artery and aortic calcification, and to improve trabecular bone mineral density in hemodialysis patients with chronic kidney disease. Here, we examined whether sevelamer given orally for 12 wk with normal food could restore bone volume (BV) and strength in aged ovariectomized (OVX) rats starting at 4 wk after OVX. Dual-energy x-ray absorptiometry, microcomputerized tomography, and bone histomorphometry analyses showed that OVX animals receiving sevelamer had increased trabecular BV (51%), trabecular number (43%), trabecular thickness (9%), cortical thickness (16%), mineral apposition rate (103%), bone formation rate (25%), and enhanced cortical and trabecular bone mechanical strength as compared with OVX rats. Sevelamer decreased collagen C telopeptide, increased osteocalcin levels, and decreased phosphate and magnesium levels without affecting calcium levels in the blood. Although sevelamer was not absorbed systemically, it stimulated osteoblast differentiation in BM-derived mesenchymal stem cell cultures, as evaluated by alkaline phosphatase positive colony-forming units, and inhibited recombinant human soluble receptor activator of nuclear factor-kappaB ligand-induced osteoclast differentiation, as evaluated by tartrate-resistant acid phosphatase positive cells in bone mineral-hematopoietic stem cell cultures. Surface enhanced laser desorption/ionization time-of-flight mass spectrometry analysis revealed that 69 proteins were differently expressed after OVX, of which 30% (20 of 69) were reversed to sham activity after sevelamer intake. PTH, fibroblast growth factor-23, and cytokine profile in serum were not significantly changed. Together, these results suggest that sevelamer in food increases the BV and improves biomechanical properties of bone in OVX rats.

The utility of using genomic DNA directly in agarose, i.e. cloneless libraries, in place of large clone libraries, radiation hybrid panels, or chromosome dissection was demonstrated. The advantage of the cloneless library approach is that, in principle, a targeted genomic resource can be developed rapidly for any genomic region using any genomic DNA sample. Here, a human chromosome 20 Not I fragment library was generated by slicing a pulsed field gel lane containing fractionating Not I cleaved DNA from a monosomic hybrid cell line into 2 mm pieces. A reliable PCR method using agarose embedded DNA was developed. InterAlu PCR generated unique patterns of products from adjacent slices (e.g. fractions). Further, the specificity of the interAlu products was demonstrated by FISH analysis and in other hybridization experiments to arrayed interAlu products. STS content mapping was used to order the fractions and also demonstrate the unique content of the library fractions.

The identification of proteins that interact with polycystin-1, the product of the autosomal dominant polycystic kidney disease gene, is an important step towards understanding the molecular pathogenesis of the disease. We have developed a two-step approach for the efficient identification of potential polycystin-1 ligands using the T7 phage display system. The first enrichment step of 4-5 rounds of biopanning is followed by a second step of reverse protein overlay assay. Thus, the sequencing efforts are minimized to the analysis of only positive rather than randomly chosen clones from the enriched population as in the standard phage display approach. Most importantly, the modified approach immediately provides the confirmation of the specificity of interaction and discriminates between strong and weak interactions. Here we present several potential interactors with distinct regions of polycystin-1, representing high-affinity binding partners.

The 14 kb mRNA of the polycystic kidney disease gene PKD1 encodes a novel large (approximately 460 kDa) protein, polycystin-1, of unknown function that is responsible for autosomal dominant polycystic kidney disease (ADPKD). The unique organization of multiple adhesive domains of polycystin-1, including 16 Ig-like domains (or PKD domains) suggests that it may play an important role in cell-cell/cell-matrix interactions. Here we demonstrate the localization of polycystin-1 to epithelial cell-cell contacts in culture. These results along with structural predictions prompted us to propose that polycystin-1 is involved in cell-cell adhesion through its cluster of Ig-like repeats. We show that Ig-like domains II-XVI are involved in strong calcium-independent homophilic interactions in vitro. Domains XI-XVI form interactions with high affinity (K(d) = 60 nM) and domains II-V exhibit the lowest binding affinity (K(d) = 730 nM) in these studies. Most importantly, we show that antibodies raised against Ig-like domains of polycystin-1 disrupt cell-cell interactions in MDCK cell monolayers, thus indicating that polycystin-1 is directly involved in the cell-cell adhesion process. Collectively, these data suggest that interactions of the Ig-like repeats of polycystin-1 play an important role in mediating intercellular adhesion. We suggest that the loss of these interactions due to mutations in polycystin-1 may be an important step in cystogenesis.

This article describes the sequence-specific isolation and purification of intact double-stranded DNA (dsDNA) by oligonucleotide/PNA-assisted affinity capture (OPAC). The OPAC assay is based on selective tagging of a DNA duplex by biotinylated oligodeoxyribonucleotide (ODN) through formation of a so-called PD-loop. The PD-loop is assembled with the aid of a pair of PNA "openers", which allow sequence-specific targeting with a Watson-Crick complementary ODN probe in the exposed region of the dsDNA. The protocol involves three steps. First, two cationic bis-PNAs locally pry the DNA duplex apart at a predetermined site. Then, the exposed DNA single strand is targeted by a complementary biotinylated ODN to selectively form a stable PD-loop complex. Finally, the capture of dsDNA is performed using streptavidin covered magnetic beads. The OPAC procedure has many advantages in the isolation of highly purified native DNA over other affinity capture and amplification techniques.

A stable complex between duplex DNA and an oligonucleotide is assembled with the aid of a DNA synthetic mimic, peptide nucleic acid (PNA). Homopyrimidine PNAs are known to invade into short homopurine tracts in duplex DNA forming P-loops. We have found that P-loops, formed at two closely located purine tracts in the same DNA strand separated by a mixed purine-pyrimidine sequence, merge and open the double helix between them. The opposite DNA strand, which is not bound with PNA, exposes and becomes accessible for complexing with an oligonucleotide via Watson-Crick pairing. As a result, the PD-loop emerges, which consists of locally open duplex DNA, PNA "openers," and an oligonucleotide. The PD-loop stability and sequence specificity are demonstrated by affinity capture of duplex DNAs by using biotinylated oligonucleotides and streptavidin-covered magnetic beads. The type of complex formed by PNAs, an oligonucleotide and duplex DNA we describe, opens ways for development of various in vitro and in situ hybridization techniques with duplex DNA and may find applications in DNA nanotechnology and genomics.

Most strains of Helicobacter pylori from patients with peptic ulcer disease or intestinal-type gastric cancer carry cagA, a gene that encodes an immunodominant protein of unknown function, whereas many of the strains from asymptomatically infected persons lack this gene. Recent studies showed that the cagA gene lies near the right end of a approximately 37kb DNA segment (a pathogenicity island, or PAI) that is unique to cagA+ strains and that the cag PAI was split in half by a transposable element insertion in the reference strain NCTC11638. In complementary experiments reported here, we also found the same cag PAI, and sequenced a 39 kb cosmid clone containing the left 'cagII' half of this PAI. Encoded in cagII were four proteins each with homology to four components of multiprotein complexes of Bordetella pertussis ('Ptl'), Agrobacterium tumefaciens ('Vir'), and conjugative plasmids ('Tra') that help deliver pertussis toxin and T (tumour inducing) and plasmid DNA, respectively, to target eukaryotic or prokaryotic cells, and also homologues of eukaryotic proteins that are involved in cytoskeletal structure. To the left of cagII in this cosmid were genes for homologues of HsIU (heat-shock protein) and Era (essential GTPase); to the right of cagII were homologues of genes for a type I restriction endonuclease and ion transport functions. Deletion of the cag PAI had no effect on synthesis of the vacuolating cytotoxin, but this deletion and several cag insertion mutations blocked induction of synthesis of proinflammatory cytokine IL-8 in gastric epithelial cells. Comparisons among H. pylori strains indicated that cag PAI gene content and arrangement are rather well conserved. We also identified two genome rearrangements with end-points in the cag PAI. One, in reference strain NCTC11638, involved IS605, a recently described transposable element (as also found by others). Another rearrangement, in 3 of 10 strains tested (including type strain NCTC11637), separated the normally adjacent cagA and picA genes and did not involve IS605. Our results are discussed in terms of how cag-encoded proteins might help trigger the damaging inflammatory responses in the gastric epithelium and possible contributions of DNA rearrangements to genome evolution.

A representative genomic library of the Corynebacterium glutamicum ATCC 13032 genes in a cosmid vector Lorist6 was created. The cosmids contain inserts of bacterial DNA obtained by partial digestion with the Sau3A I restrictase. Five hundred and thirty individual primary recombinant clones were transferred into the wells of microtiter plates, where they are now being preserved. The average size of the bacterial DNA inserts determined via a sum of restriction fragment sizes of recombinant molecules is about 38 kb. The capacity of the obtained gene library is 8.4 equivalents of the C. glutamicum genome, i.e., every fragment of the genome is on average represented by eight clones and is presented in at least one clone with the probability > 99%. Clone grids (sets of recombinant clones located on the hybridization membrane in regular and reproducible order) were created. Specificity of the created clone library and its representativeness were confirmed experimentally by hybridization of clone grids with DNA probes corresponding to unique regions of the Corynebacterium genome. A plasmid containing the pheA prephenate dehydratase gene, olygonucleotide corresponding to the lysC gene, and the 21 RNA probe obtained from the insert ends in different cosmids were used as probes. The created set of clones allows the construction of a cosmid contig overlapping the C. glutamicum genome and a physical genetic map on its base.

Conventional genome mapping and sequencing involves the analysis and processing of individual samples and pieces of experimental data. Although these methods work, it is quite clear that more efficient and less expensive methods are needed. Our top down physical mapping experiments have focused on the parallel processing of information from multiple samples at one time. This approach has aided the construction of genomic restriction maps and allowed us to assess the degree of large-scale conservation across wide regions of the human genome. The principles of parallel processing were applied in top down experiments that ordered an overlapping cosmid library from the 14-Mb Schizosaccharomyces pombe genome. This approach produced an eight-fold increase in efficiency in clone ordering over similar efforts. Recently, we have developed an enhanced sequencing by hybridization protocol that allows DNA sequence information to be collected on a large number of samples at once. Our current research focuses on applying parallel processing principles to make genome-wide comparisons between pairs of samples for analyzing disease states.

Infection of the eye by Pseudomonas aeruginosa can result in corneal inflammation (keratitis) and ulceration, and permanent decrease in vision if not successfully treated. We tested for diversity among P. aeruginosa strains from corneal ulcers by the sensitive and efficient 'RAPD' (for 'random amplified polymorphic DNA') fingerprinting method. This method uses single oligonucleotides of arbitrarily chosen sequence as primers in low-stringency PCR amplification, and results in strain-specific arrays of DNA fragments. Tests of 20 independent P. aeruginosa corneal ulcer isolates yielded 19 different arrays of products with each of three arbitrary primers, indicating that all but two of the strains differed from one another. Additional isolates from three patients (infected eye, contact lens or eye drops) yielded fragment patterns that were identical to those of the original isolate in each case. Thus, our results demonstrate considerable diversity among P. aeruginosa corneal ulcer isolates, and suggest that just one clone may predominate in typical infections.

Helicobacter pylori is a Gram-negative bacterium that infects the human gastric mucosa, causes gastritis and contributes to the development of peptic ulcers and gastric cancer. To facilitate molecular genetic analysis of this pathogen, we constructed a approximately 20-fold redundant cosmid library and physical/genetic map of strain NCTC11638. Genomic DNA fragments were cloned into the cosmid vector Lorist6, and clones were ordered by hybridization with several types of probes: (i) ends of cloned DNAs; (ii) chromosomal Notl digest fragments; (iii) cosmids containing Notl sites; and (iv) specific genes. Seven hundred and fifty-one cosmids were mapped to one of three contigs covering > 90% of the chromosome, and are represented by a 68-cosmid miniset. The order of cosmids was confirmed and extents of overlap among them were estimated by restriction analysis. All currently known H. pylori genes were mapped, including those for a cytotoxin (vacA), cytotoxin-associated protein (cagA), urease and regulatory functions (ureAb, ureD and ureH), catalase (katA), major and minor flagellins (flaA and flaB), heat-shock (stress) and chaperone proteins (dnaK, htA, hspB (groEL)), prokaryotic ferritin (pfr), an adhesin subunit (hpaA), a surface protein (26 kDa), and 16S and 23S ribosomal RNAs (two genes each). The orientations of eight genes or clusters were determined, and two repetitive sequences were also found. The gene order and rRNA gene copy number determined here differed from that reported for an unrelated strain, which suggests considerable flexibility in H. pylori genome organization.

DNA sequence diversity among 60 independent isolates of the gastric pathogen Helicobacter pylori was assessed by testing for restriction fragment length polymorphisms (RFLPs) in several PCR-amplified gene segments. 18 Mbol and 27 HaeIII RFLPs were found in the 2.4 kb ureA-ureB (urease) segment from the 60 strains; this identified 44 separate groups, with each group containing one to four isolates. With one exception, each isolate not distinguished from the others by RFLPs in ureA-ureB was distinguished by Mbol digestion of the neighboring 1.7 kb ureC-ureD segment. The 1.5 kb flaA (flagellin) gene, which is not close to ure gene cluster, was also highly polymorphic. In contrast, isolates from initial and followup biopsies yielded identical restriction patterns in each of the three cases tested. The potential of this method for detecting population heterogeneity was tested by mixing DNAs from different strains before amplification: the arrays of restriction fragments obtained indicated co-amplification from both genomes in each of the five pairwise combinations tested. These results show that H. pylori is a very diverse species, that indicate PCR-based RFLP tests are almost as sensitive as arbitrary primer PCR (RAPD) tests, and suggest that such RFLP tests will be useful for direct analysis of H. pylori in biopsy and gastric juice specimens.

The RAPD (or AP-PCR) DNA fingerprinting method was used to distinguish among clinical isolates of Helicobacter pylori, a bacterium whose long term carriage is associated with gastritis, peptic ulcers and gastric carcinomas. This method uses arbitrarily chosen oligonucleotides to prime DNA synthesis from genomic sites to which they are fortuitously matched, or almost matched. Most 10-nt primers with > or = 60% G + C yielded strain-specific arrays of up to 15 prominent fragments, as did most longer (> or = 17-nt) primers, whereas most 10-nt primers with 50% G+C did not. Each of 64 independent H. pylori isolates, 60 of which were from patients in the same hospital, was distinguishable with a single RAPD primer, which suggests a high level of DNA sequence diversity within this species. In contrast, isolates from initial and followup biopsies were indistinguishable in each of three cases tested.

Plasmids carrying an asd gene from a mutant. S-(2-aminoaethyl)-L-cysteine resistant strain of Corynebacterium glutamicum were selected from a clonoteque constructed on a plasmid cloning vector pSL5 by complementation of asd mutation in Escherichia coli. Evidence has been obtained that the cloned chromosomal DNA fragment contains also a complete sequence for feed-back-resistant aspartokinase lysC gene.

Novel cloning vectors for glutamic acid producing bacteria have been constructed. The cryptic plasmid pBO1 (4.4 kb) from Brevibacterium sp. recombined with the plasmid pACYC184 (4.0 kb) from Escherichia coli was used to produce composite plasmid named pKA1. The plasmid could propagate and express the Cm-r phenotype in E. coli and coryneform glutamic acid producing bacteria Br. flavum, C. glutamicum, Br. lactofermentum. The pKA1 plasmid and its variants deleted within non-essential plasmid regions with unique restriction sites HindIII, SalGI, SphI were used in cloning experiments. The genes coding for threonine biosynthesis of C. glutamicum and Br. flavum were subcloned into shuttle vectors in C. glutamicum cells. Recombinant plasmids were introduced into protoplasts by polyethylenglycol-mediated transformation of plasmid DNAs. It was shown that the presence of plasmids containing the Br. flavum thrA2 gene in C. glutamicum (thrB) caused 10-fold increase in homoserine dehydrogenase activity, as compared to that of wild type strain, and in homoserine production.

A library of EcoRI DNA fragments from Brevibacterium flavum was constructed using plasmid vector. The genes complementing ThrA2 and ThrB mutations in Escherichia coli were identified in the library. The gene thrA2 of B. flavum codes for mutant enzyme homoserine dehydrogenase insensitive to inhibition by threonine. The genes thrA2 and thrB are localized wihtin the EcoRI fragment 4.1 kb long and are expressed under the control of their own promoters in E. coli cells. Structural and functional analysis of cloned C. glutamicum gene ilvA was performed. The gene of C. glutamicum complemented ilvA mutation in E. coli and appeared to be localized within the EcoRI--SacI DNA fragment 1.6 kb in size. Using E. coli minicells we have demonstrated that the gene ilvA of C. glutamicum controls the synthesis of polypeptide of relative molecular mass 50 kD.

Pectate lyase synthesis in the cells of Erwinia carotovora ELA 49 is induced by polypectate. This suggested that the Erwinia chromosomes carried a regulator gene responsible for negative regulation of the pectate lyase gene expression. In the present study the regulator gene controlling expression of one of the pectate lyase structural genes was cloned and designated as ptlA gene. For this purpose a genetic system with the tester plasmid pPc624 as the main element was constructed. The tester plasmid contained cat gene (resistance to chloramphenicol) controlled by the promotor of the ptlA gene cloned on vector pPD620. Plasmid pPC624 was maintained in the E. coli cells in a number of 1-2 copies and transferred resistance to chloramphenicol in concentrations up to 100 micrograms/ml to the cells. The E. carotovora cells containing pPC624 were sensitive to chloramphenicol in media containing no inductor (sodium polypectate). In media with the inductor they were resistant to chloramphenicol. Therefore, plasmid pPC624 proved to be a suitable system for testing the regulator gene product. The E. coli cells containing plasmid pPC624 were transformed by the hybrid Ptl+ plasmids identified in the clonotheque of the Erwinia DNA EcoRI fragments. The E. coli cotransformants were characterized by chloramphenicol sensitivity which provided a conclusion that the regulator ptlR gene controlling the ptlA gene expression was localized on the DNA EcoRI fragment (7.3 kb) containing the pectate lyase ptlA and ptlB genes. Deletion analysis showed that the investigated genes were localized in the EcoRI fragment (7.3 kb) of the E. carotovora chromosomal DNA in the following order: ptlA--ptlB--ptlR.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning and expression of Corynebacterium glutamicum genes complementing Escherichia coli mutations thrA2 and ilvA was performed. It was demonstrated that the thrA2 gene of C. glutamicum is located close to thrB on EcoRI DNA fragment 4.1 kb long. The fragment was cloned in pUC18 vector. The thrA2 gene is expressed in the recombinant plasmid pOBT3 under control of the vector pUC18 Plac promoter. In E. coli minicells, the genes thrA2 and thrB determined synthesis of proteins of Mr 43kD and 25 kD, respectively. A gene complementing ilvA mutation of E. coli was identified in a library of EcoRI C. glutamicum DNA fragments. This library was constructed using plasmid vector. It was shown that the ilvA gene of C. glutamicum is located inside the 3.6 kb EcoRI fragment and is expressed using its own promoter.

Phasmid lambda pMYF131, a hybrid of phage lambda vectors and plasmid pUC19, was constructed. The phasmid and its derivatives were shown to be efficient vectors for construction and analysis of gene libraries in Escherichia coli cells. The lambda pMYF131 DNA molecule contains all the genes and regions essential for phage lytic development. The plasmid cannot be packaged either in the monomeric or the oligomeric form due to its specific length. Elongation of the DNA molecule by ligation with fragments of foreign DNA can make it packageable and this is easily detected by plaque formation. Hence, the procedures used to construct genomic libraries can be simplified by selection of only recombinant DNA molecules just at the time and on the basis of their packaging in vitro. The output of recombinant clones per vector molecule was several times higher for vector lambda pMYF131, compared to phage vector lambda L47.1AB, and attained 3 x 10(6) clones per micrograms DNA. Vector and recombinant phasmids can be obtained in large quantities in plasmid form. lambda pMYF131 contains nine unique restriction sites which allow the cloning of DNA fragments with blunt ends and of fragments with various types of cohesive ends, obtained by digestion with 14 prototype restriction enzymes. The maximal size of the cloned DNA fragments is approx. 20 kb for lambda pMYF131. Phasmid vectors were used to construct libraries of bovine, pig and quail genomes, and genomic libraries of 17 species of bacteria. Application of suitable methods allowed the identification 13 individual genes within these libraries.

The 69-6 strain of Bacillus thuringiensis subsp. galleriae harbours at least 7 cryptic plasmids (pBTG1 - pBTG7) with molecular lengths 8,4 to 15,7 kb. According to hybridization analysis, the plasmid pBTG2 (8,7 kb) and other plasmids of the same host strain as well as cryptic plasmids of the strains belonging to 10 other serotypes of Bac. thuringiensis share detectable homology. As shown by the data of heteroduplex analysis, about 60% of pBTG1 and pBTG2 genomes have homologous DNA sequences. These data point out tht some plasmid genes are conserved in Bac. thuringiensis. BasmHI-, EcoRI- and HindIII-generated fragments of Bac. thuringiensis subsp. galleriae strain 69-6 are cloned on the pBR325 vehicle in the cells of Escherichia coli.

Erwinia chrysanthemi ENA49 structural and regulatory ptl genes, coding for pectate lyase (Ptl) were cloned in Escherichia coli cells. Phage vector lambda L47.1 and phasmid vector lambda pMYF131 were used for constructing libraries of BamHI and EcoRI fragments, respectively, of Er. chrysanthemi chromosomal DNA. Among the 1,100 hybrid clones containing BamHI Er. chrysanthemi DNA fragments and 11,000 hybrid clones containing EcoRI fragments, six and 45 clones, respectively, were identified as having pectolytic activity. Two different structural genes, designated ptlA and ptlB, have been subcloned on multi-copy plasmids. Genes ptlA and ptlB are located side by side on the chromosome of Er. chrysanthemi and transcribe in the same direction. Each of the genes has its own promoter. Southern-blot hybridization analysis showed that the cloned ptl genes shared practically no homology and each of the genes was represented by a single copy on the Er. chrysanthemi chromosome. Other ptl genes capable of expression in E. coli cells were not found in the gene libraries. Negative regulation of the ptlA gene expression by a cloned gene called ptlR was shown. To screen the gene library for the ptlR gene, a specific genetic system was devised. The genes studied are located within an EcoRI chromosomal DNA fragment of 7.3 kb in the order: ptlA-ptlB-ptlR.