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Biomedical subjects

S Saadi

Publications and source records attributed to S Saadi.

33 records · Page 2Linked to original sources
PubMed1991

Differentiation of lethal and nonlethal, kor-regulated functions in the kilB region of broad host-range plasmid RK2.

In broad host-range plasmid RK2, several kil loci (kilA, kilB, kilC, kilE) and the replication initiator gene (trfA) are regulated by combination of kor determinants (korA, korB, korC, korE) in a regulatory network known as the kil-kor region. Although the kil determinants are not essential for replication, their coregulation with trfA suggests an involvement in plasmid maintenance or host-range. Plasmids carrying the cloned kilB region of RK2 cannot be maintained in the absence of korB owing to two phenotypically distinguishable, kor-regulated determinants: (1) kilB1 (kilD), which can be controlled by korA or korB, and (2) kilB2, which requires korB for control. In this study, we have determined the nature of the functions responsible for the kor-sensitive phenotypes of the kilB region. We found that insertion of transcription terminators within or downstream of the trfA operon allows plasmids carrying the kilB1 portion of the kilB region to be maintained in cells lacking korA or korB. In addition, mutants of the kilB1 region that can be maintained in the absence of korA and korB have alterations in the trfA promoter. These results show that the phenotype of the cloned kilB1 region in kor-deficient cells depends on trfA transcription but does not involve expression of any gene of the trfA operon. Therefore, the kilB1 determinant is not a structural gene. The phenotype results from entry of trfA-initiated transcription into adjacent sequences of the plasmid vector. The ability to block the kilB2 phenotype with transcriptional terminators allowed us to show conclusively that the kilB2 determinant is a host-lethal gene (klbA) whose regulation is dependent on korB. These findings have implications for the structure of the basic replicon of RK2.

Base Sequence↗
PubMed1991

Isolation and properties of the RepA1 protein of the IncFII replicon, RepFIC.

The initiator protein RepA1 of the IncFII replicon RepFIC derived from the enterotoxin plasmid EntP307 has been cloned under the control of the lambda PL promoter. This has enabled us to overproduce this protein and study its properties. Here we show that RepA1 is a soluble basic protein with an experimentally determined molecular weight of 40,000. Deletion analysis indicates that the overproduced protein originates from the open reading frame which we previously designated as coding for RepA1. We have also shown that the replication function of the replicon RepFIC depends on the intact RepA1 coding frame.

Amino Acid Sequence↗
PubMed1991

Structural, molecular, and genetic analysis of the kilA operon of broad-host-range plasmid RK2.

The kil loci (kilA, kilB, kilC, and kilE) of incompatibility group P (IncP), broad-host-range plasmid RK2 were originally detected by their potential lethality to Escherichia coli host cells. Expression of the kil determinants is controlled by different combinations of kor functions (korA, korB, korC, and korE). This system of regulated genes, known as the kil-kor regulon, includes trfA, which encodes the RK2 replication initiator. The functions of the kil loci are unknown, but their coregulation with an essential replication function suggests that they have a role in the maintenance or host range of RK2. In this study, we have determined the nucleotide sequence of a 3-kb segment of RK2 that encodes the entire kilA locus. The region encodes three genes, designated klaA, klaB, and klaC. The phage T7 RNA polymerase-dependent expression system was use to identify three polypeptide products. The estimated masses of klaA and klaB products were in reasonable agreement with the calculated molecular masses of 28,407 and 42,156 Da, respectively. The klaC product is calculated to be 32,380 Da, but the observed polypeptide exhibited an apparent mass of 28 kDa on sodium dodecyl sulfate-polyacrylamide gels. Mutants of klaC were used to confirm that initiation of translation of the observed product occurs at the first ATG in the klaC open reading frame. Hydrophobicity analysis indicated that the KlaA and KlaB polypeptides are likely to be soluble, whereas the KlaC polypeptide was predicted to have four potential membrane-spanning domains. The only recognizable promoter sequences in the kilA region were those of the kilA promoter located upstream of klaA and the promoter for the korA-korB operon located just downstream of a rho-independent terminatorlike sequence following klaC. The transcriptional start sites for these promoters were determined by primer extension. Using isogenic sets of plasmids with nonpolar mutations, we found that klaA, klaB, and klaC are each able to express a host-lethal (Kil+) phenotype in the absence of kor functions. Inactivation of the kilA promoter causes loss of the lethal phenotype, demonstrating that all three genes are expressed from the kilA promoter as a multicistronic operon. We investigated two other phenotypes that have been mapped to the kilA region of RK2 or the closely related IncP plasmids RP1 and RP4: inhibition of conjugal transfer of IncW plasmids (fwB) and resistance to potassium tellurite. The cloned kilA operon was found to express both phenotypes, even in the presence of korA and korB, whose functions are known to regulate the kilA promoter. In addition, mutant and complementation analyses showed that the kilA promoter and the products of all three kla genes are necessary for expression of both phenotypes. Therefore, host lethality, fertility inhibition, and tellurite resistance are all properties of the kilA operon. We discuss the possible role of the kilA operon for RK2.

Amino Acid Sequence↗
PubMed1989

Properties and incompatibility behavior of miniplasmids derived from the bireplicon plasmid pCG86.

Many plasmids belonging to the F incompatibility groups contain more than one basic replicon. The chimeric plasmid pCG86 is an example of such a multireplicon plasmid. The two basic replicons of pCG86, RepFIIA/FIC and RepFIB have been cloned and re-ligated, the copy numbers of the clones have been determined, and the incompatibility behavior of plasmids containing the ligated replicons and the individual replicons has been studied. The bireplicon plasmids are not expected to be incompatible as recipients with monoreplicon RepFIB or RepFIIA/RepFIC plasmids, since when one replicon is challenged by an incoming replicon, the other should be able to handle the plasmid's replication. In our studies, we found that challenge with either monoreplicon plasmid resulted in incompatibility. This incompatibility was increased in bireplicon plasmids in which RepFIB was duplicated. We conclude that in the bireplicon plasmids, challenging the replication control of one replicon by an incompatible plasmid can interfere with the replication originating from the second replicon.

Cloning, Molecular↗
PubMed1988

A basic replicon of virulence-associated plasmids of Shigella spp. and enteroinvasive Escherichia coli is homologous with a basic replicon in plasmids of IncF groups.

Shigella species and enteroinvasive Escherichia coli strains carry a large (120- to 140-megadalton) plasmid called pINV, which contains genes essential for the invasiveness of these pathogens. Hybridization with specific probes derived from the RepFIC and RepFIB replicons of the IncF1 Ent plasmid P307 showed that pINVs present in 35 clinical isolates are homologous with RepFIC but not RepFIB, regardless of the serogroup of the Shigella or E. coli strain. RepFIC of P307, in turn, is very similar to RepFIIA replicons of IncFII R plasmids. These and other related replicons constitute the RepFIIA family. With one pINV, pWR110, a plasmid of Shigella flexneri 5, we demonstrated the existence of a functional replicon, RepINV, with a restriction map similar to that of RepFIIA of plasmid R1. We isolated the putative inc RNA coding region of RepINV, which is a major determinant of incompatibility. The nucleotide sequence of the RepINV-inc RNA-coding region was determined and compared with the corresponding sequences of RepFIC and RepFIIA. The differences were small, but apparently were sufficient to affect the target specificity of the inc RNAs, thus rendering the replicons compatible with each other. We conclude that pINVs present in Shigella spp. and enteroinvasive E. coli constitute a homogeneous group, containing one basic replicon that belongs to the RepFIIA family of replicons.

Base Sequence↗
PubMed1987

Nucleotide sequence analysis of RepFIC, a basic replicon present in IncFI plasmids P307 and F, and its relation to the RepA replicon of IncFII plasmids.

RepFIC is a basic replicon of IncFI plasmid P307 which is located within a 3.09-kilobase SmaI fragment. The nucleotide sequence of this region has been determined and shown to be homologous with the RepFIIA replicon of IncFII plasmids. The two replicons share three homologous regions, HRI, HRII, and HRIII, which are flanked by two nonhomologous regions, NHRI and NHRII. A comparison of coding regions reveals that the two replicons have several features in common. RepFIC, like RepFIIA, codes for a repA2 protein with its amino-terminal codons in HRI and its carboxy-terminal codons in NHRI. Although the codons for the repA1 proteins are located in NHRII, the DNA region containing a putative promoter, ribosomal binding site, and initiation codons is located in HRII. This region also codes for an inc RNA. There are nine base-pair differences between the inc RNA of RepFIIA and that of RepFIC, and as a result, RepFIC and RepFIIA replicons are compatible. An EcoRI fragment from the F plasmid which shows homology with RepFIC of P307 has also been sequenced. This fragment contains only a portion of RepFIC, including the genes for the putative repA2 protein and inc RNA. The region coding for a putative repA1 protein is interrupted by the transposon Tn1000 and shows no homology with the repA1 region of RepFIIA and RepFIC of P307. Our comparative and structural analyses suggest that RepFIC and RepFIIA, although different, have a similar replication mechanism and thus can be assigned to the same replicon family, which we designate the RepFIIA family.

Base Sequence↗
PubMed1986

Distribution of basic replicons having homology with RepFIA, RepFIB, and RepFIC among IncF group plasmids.

Plasmids encoding F-like pili have been divided into groups on the basis of their incompatibility behavior. Three basic replicons have been recognized previously in the IncFI plasmid group and we have now examined their distribution in representative plasmids from 22 of the currently recognized incompatibility groups. The occurrence of these basic replicons was found to be rare outside of the IncF group, and significant hybridization was shown only for RepFIA to IncH1 and I group plasmids. Homology to the RepFIC basic replicon was found in all but one of the IncF group plasmids examined but RepFIA and RepFIB have a more restricted distribution. It appears likely that some plasmids carry vestiges of replicons which still express incompatibility but are incapable of replication. We suggest that evolutionary divergence among the plasmids of the IncF group has resulted from various genetic rearrangements among these basic replicons.

Cloning, Molecular↗
PubMed1984

Characterization of the basic replicons of the chimeric R/Ent plasmid pCG86 and the related Ent plasmid P307.

Restriction-enzyme fragments that can replicate autonomously after circularization were isolated from the chimeric R/Ent plasmid pCG86 and the Ent plasmid P307. Two such regions containing a basic replicon were located in each plasmid. One of the basic replicons of P307, RepFIB, is almost identical with one of the basic replicons of pCG86. The other basic replicon in P307, RepFIC, is partly homologous with the second basic replicon in pCG86, RepFIIA/RepFIC. The latter is a hybrid basic replicon and is in addition partly homologous with RepFIIA, a basic replicon present in IncFII R plasmids. By restriction-enzyme mapping and nucleotide-sequence analysis we have determined a site in the hybrid replicon where it ceases to be homologous with the RepFIIA basic replicon contained in the IncFII miniplasmid pSM1. The 2410-bp region of homology with pSM1 corresponds with a segment containing the origin of replication and all the genes responsible for replication control of pSM1.

Base Sequence↗
PubMed1984

RepFIC, a basic replicon of IncFI plasmids that has homology with a basic replicon of IncFII plasmids.

It was found that a DNA segment containing genes for autonomous replication and its control (basic replicon) present in the IncFI plasmid P307 has homology with RepA, a basic replicon present in IncFII plasmids. The basic replicon in P307 is referred to as RepFIC and the homologous basic replicon in IncFII plasmids is referred to as RepFIIA. In 11 other IncFI plasmids studied a region that has homology with RepFIC and RepFIIA was demonstrated. Thus, of the several basic replicons present in IncFI plasmids, RepFIC is evolutionarily related to a basic replicon of IncFII plasmids.

Base Sequence↗
PubMedDate not supplied

The role of complement in transplantation.

The complement system contributes critically to the barrier to transplantation of cells and organs. In the case of tissues and organs transplanted between individuals of the same species, that is in allotransplantation, the barrier posed by complement is seemingly eclipsed by the barrier posed by cellular immune responses. In the case of cells and organs transplanted between individuals of disparate species, that is xenotransplantation, the complement system has been thought to pose a nearly insurmountable barrier. With our understanding on how the complement system contributes to rejection, it is now clear that the complement system is more important in allotransplantation and more forgiving in xenotransplantation than was previously thought.

Acute Disease↗
PubMedDate not supplied

[Progressive familial intrahepatic cholestasis (Byler disease): current genetics and therapy].

Progressive familial intrahepatic cholestasis (PFIC) is a congenital liver disease. First symptoms can frequently be seen shortly after birth. Quality and expectation of life are substantially reduced due to severe pruritus and the complications of progressive liver cirrhosis. PFIC is diagnosed on the basis of characteristic clinical and laboratory parameters and genetic analysis after exclusion of other liver diseases leading to intrahepatic cholestasis. Medical therapy is only effective in a proportion of children with PFIC. Partial biliary diversion (PBD) is nowadays considered the therapy of choice in patients with therapy-refractive pruritus. If performed in time, damage to the liver can be delayed or arrested, thus orthotopic liver transplantation (OLT) can be postponed or even avoided in at least some patients with PFIC. Besides providing a current overview of PFIC, we report on three patients who were successfully treated surgically. One patient was subjected to a new technique of PBD (cholecysto-appendicostomy), the other two had OLT.

Appendix↗
PubMedDate not supplied

Iron-deficient medium for selective isolation and presumptive identification of enterococci.

An Enterococcus Selective Agar (ESA; basal medium plus 0.01% NaN3, pH 9.6, Fe ion-restricted) was applied to a direct, single-step, isolation and presumptive identification of enterococci from clinical urine. The ESA was examined as to rate of colony formation and selectivity together with two conventional culture media, Pfizer Selective Enterococcus Agar (PSE) and Phenyl Ethyl Alcohol Agar (PEA). Significant differences were observed in the rate of colony formation after 14, 17, and 20 hr of incubation at 35 degrees. The size of colonies on ESA was consistently larger than on the other two media. Biochemical testing showed that isolates from ESA conformed more closely to the definition of enterococci than isolates from either PSE or PEA. The improved selectivity of ESA was in large part due to the reduced presence of Fe ions. The medium offers advantages for an expeditious and accurate isolation of enterococcal pathogens.

Agar↗