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The occurrence of membranoproliferative glomerulonephritis in a 13 year old boy with inherited complete deficiency of the second component of complement (C2) is described here for the first time. Results of the complement studies and the associations of glomerulonephritis with complement deficiencies are discussed.
The prgB gene encodes the surface protein, Asc10, which mediates cell aggregation, resulting in high-frequency conjugative transfer of the pheromone-inducible tetracycline-resistance plasmid pCF10 in Enterococcus faecalis. Messenger RNA analysis by Northern blot hybridization and primer extension indicates that prgB transcription is pheromone-inducible and monocistronic. Previous transposon mutagenesis and sequencing analysis of a 12-kilobase (kb) region of pCF10 indicated that several genes including prgR and prgS are required to activate expression of prgB. The distance (3-4 kb) between these regulatory genes and prgB suggested that the activation might function in trans. To test this, a promoterless lacZ gene fusion to prgB was constructed and cloned without some or all of the regulatory genes. Several restriction fragments of the regulatory region were cloned in a higher copy-number plasmid, and numerous complementation studies were carried out in E. faecalis. Complementation in trans was not observed in any of these experiments. However, when the regulatory region and target genes were cloned in different sites of the same plasmid, separated by as much as 12 kb, activation of prgB was observed. Interestingly, this activation occurred only when the regions were cloned in the same relative orientation in which they exist on wild-type pCF10. These results suggest that one or more regulatory molecules may bind to an upstream cis-acting site and track along the DNA to reach a target site to activate prgB transcription.
Cell lines were generated by cotransfection of Vero cells with pSV2neo and a plasmid containing the herpes simplex virus type 1 (HSV-1) EcoRI D fragment (coordinates 0.086 to 0.194). One such cell line (S22) contained the genes for alkaline exonuclease and several uncharacterized functions. Three mutant isolates of HSV-1 strain KOS which grew on S22 cells but not on normal Vero cells were isolated and characterized. All three mutants (hr27, hr48, and hr156) were defective in the synthesis of viral DNA and late proteins when grown in nonpermissive Vero cells. Early gene expression in cells infected with these host range mutants appeared to be normal at the nonpermissive condition. The mutations were mapped by marker rescue to a 1.5-kilobase fragment (coordinates 0.145 to 0.155). The mutation of one of these mutants, hr27, was more finely mapped to an 800-base-pair region (coordinates 0.145 to 0.151). This position of these mutations is consistent with the map location of a putative 94-kilodalton polypeptide as determined by sequence analysis (D. McGeoch, personal communication). Complementation studies demonstrated that these mutants formed a new complementation group, designated 1-36. The results presented in this report indicate that the 94-kilodalton gene product affected by these mutations may have a direct role in viral DNA synthesis.
Tn2501 is a cryptic class II transposon found as part of the lactose transposon Tn951. Insertional inactivation and nucleotide sequence analysis of Tn2501 allowed us (i) to localize the transposase (tnpA) and the resolvase (tnpR) genes as well as the resolution site (res) of Tn2501 and (ii) to compare Tn2501 with other well-known elements of the two subgroups of class II transposons (Tn3, gamma delta, Tn951, IS101; and Tn21, Tn501, Tn1721). The genetic organization of Tn2501 is similar to that of Tn3 with divergent transcription of the tnpA and tnpR genes away from the intervening res site. The tnpR gene of Tn2501 shows weak homology with that of Tn3 and even less with those of Tn21 and Tn501. However, the tnpA gene and the inverted repeat sequences of Tn2501 present more homology with those of Tn21 and Tn501 than with those of Tn3. Complementation studies showed that TnpA- mutants of Tn2501 can be complemented, at a low frequency, by the Tn21 transposase. None of the tested transposons complemented TnpR- mutants of Tn2501.
To identify nuclear functions required for cytochrome c oxidase biogenesis in yeast, recessive nuclear mutants that are deficient in cytochrome c oxidase were characterized. In complementation studies, 55 independently isolated mutants were placed into 34 complementation groups. Analysis of the content of cytochrome c oxidase subunits in each mutant permitted the definition of three phenotypic classes. One class contains three complementation groups whose strains carry mutations in the COX4, COX5a, or COX9 genes. These genes encode subunits IV, Va, and VIIa of cytochrome c oxidase, respectively. Mutations in each of these structural genes appear to affect the levels of the other eight subunits, albeit in different ways. A second class contains nuclear mutants that are defective in synthesis of a specific mitochondrial-encoded cytochrome c oxidase subunit (I, II, or III) or in both cytochrome c oxidase subunit I and apocytochrome b. These mutants fall into 17 complementation groups. The third class is represented by mutants in 14 complementation groups. These strains contain near normal amounts of all cytochrome c oxidase subunits examined and therefore are likely to be defective at some step in holoenzyme assembly. The large number of complementation groups represented by the second and third phenotypic classes suggest that both the expression of the structural genes encoding the nine polypeptide subunits of cytochrome c oxidase and the assembly of these subunits into a functional holoenzyme require the products of many nuclear genes.
Animals were depleted of complement using cobra venom factor and the influence of complement depletion on the course of renal infection was studied. Complement depletion markedly increased the susceptibility of renal tissue to a challenge with an E. coli strain sensitive to the bactericidal activity of normal serum, but did not influence the outcome of a challenge with a serum-resistant strain of E. coli. These observations are consistent with the hypothesis that complement-mediated host immune mechanisms do play a role in the biology of renal infection and are an important component of the host's immune response in pyelonephritis.
The history of the peroxisomal disorders (PDs), including the most frequent variant, the cerebrohepatorenal syndrome of Zellweger, can be divided into four phases. During the first phase, lasting from 1964 to 1972, the clinical and pathologic manifestations of Zellweger's syndrome (ZS) were explored and delineated. In 1973 it was found that ZS is due to the absence of peroxisomes in hepatocytes and renal tubular epithelial cells. With this discovery the second phase of ZS was initiated, which in subsequent years led to discovery of various defective peroxisomal functions. During the third phase, beginning in 1980, various other peroxisomal disorders were discovered, among them infantile Refsum's disease, hyperpipecolic acidemia, neonatal adrenoleukodystrophy, and rhizomelic chondrodysplasia punctata. During 1986 the etiology of the various PDs was identified by complementation studies, marking the beginning of the fourth phase of the history of the peroxisomopathies. It was found that ZS, neonatal adrenoleukodystrophy, and rhizomelic chondrodysplasia punctata represent different genetic entities, while Refsum's disease and hyperpipecolic acidemia are alleviated variants of ZS. Moreover, results of preliminary studies indicate that cells of one case of ZS may complement the cells of another ZS case, which could indicate genetic heterogeneity of ZS.
We have characterized a SV40-transformed human fibroblast cell line (GM6914) derived from a patient with Fanconi anemia (FA) in order to establish its usefulness for biochemical and genetic experiments, including DNA-mediated gene transfer. GM6914 cells have a growth rate similar to that of SV40-transformed normal human fibroblasts and an indefinite lifespan in culture. As has been established for other FA cell types, GM6914 cells have an increased sensitivity to DNA-crosslinking agents such as mitomycin C (MMC). The D10 for GM6914 cells is 8 times lower than for equivalent controls. GM6914 cells also have an elevated frequency of spontaneous chromosome aberrations and this frequency can be increased by MMC concentrations which show no effect on control cells. Genetic complementation studies with lymphoblasts derived from two affected sibs of the donor of GM6914 cells show that GM6914 belongs to FA complementation group A. In DNA-transfection studies using plasmid pRSVneo, colonies of GM6914 cells resistant to the drug G-418 were observed at frequencies ranging from 1.7 to 16 X 10(-4), values similar to those observed with several other SV40-transformed human cell lines. GM6914 should be a useful recipient cell line in experiments using DNA-mediated gene transfer to clone the normal allele of the gene which is defective in FA complementation group A. GM6914 would also be an excellent cell line for studies on mutagenesis, recombination and repair using plasmid vectors.
The genetic locus glt, encoding glutamate synthase from Rhizobium meliloti 1021, was selected from a pLAFR1 clone bank by complementation of the R. meliloti 41 Glt- mutant AK330. A fragment of cloned DNA complementing this mutant also served to complement the Escherichia coli glt null mutant PA340. Complementation studies using these mutants suggested that glutamate synthase expression requires two complementation groups present at this locus. Genomic Southern analysis using a probe of the R. meliloti 1021 glt region showed a close resemblance between R. meliloti 1021, 41, and 102f34 at glt, whereas R. meliloti 104A14 showed many differences in restriction fragment length polymorphism patterns at this locus. R. meliloti 102f34, but not the other strains, showed an additional region with sequence similarity to glt. Insertion alleles containing transposable kanamycin resistance elements were constructed and used to derive Glt- mutants of R. meliloti 1021 and 102f34. These mutants were unable to assimilate ammonia and were Nod+ Fix+ on alfalfa seedlings. The mutants also showed poor or no growth on nitrogen sources such as glutamate, aspartate, arginine, and histidine, which are utilized by the wild-type parental strains. Strains that remained auxotrophic but grew nearly as well as the wild type on these nitrogen sources were readily isolated from populations of glt insertion mutants, indicating that degradation of these amino acids is negatively regulated in R. meliloti as a result of disruptions of glt.
Kupffer cells are well known to be important for normal host defense function. The development of methods to evaluate the in vivo function of specific receptors on Kupffer cells has made it possible to assess the role of these receptors in host defense. The rationale for studying complement receptors is based on the proposed important role of these receptors in host defense and on the observation that the hereditary deficiency of a complement receptor is associated with recurrent severe bacterial infections. The studies reviewed here demonstrate that forms of injury that are associated with depressed host defense including thermal injury, hemorrhagic shock, trauma, and surgery also cause a decrease in complement receptor clearance function. This decrease in Kupffer cell receptor clearance function was shown not to be the result of depressed hepatic blood flow or depletion of complement components. Complement receptor function was also depressed following the phagocytosis of particulates that are known to depress Kupffer cell host defense function. Endotoxemia and bacteremia also were associated with a depression of complement receptor function. Complement receptor function was experimentally depressed in uninjured animals by the phagocytosis of IgG-coated erythrocytes. There was a close association between the depression of complement receptor clearance function and increased susceptibility to the lethal effects of endotoxin and bacterial infection. These studies support the hypotheses that complement receptors on Kupffer cells are important for normal host defense and that depression of the function of these receptors impairs host defense.
Various tumours of the urogenital system were studied whether their cells contain gene products characteristic of functioning adenovirus genes. Complementation studies were made by using tumour cell extracts and two temperature-sensitive adenovirus mutants at restrictive and permissive temperatures. The procedure was applied in in vitro HEp-2 cell culture. It was found that cell extracts of bladder tumour, renal tumour, malignant bladder papilloma and of penis cancer are capable of complementing adenovirus mutants at restrictive temperature, i.e. they induce the reproduction of virus mutants being otherwise incapable of replication. Thus, functioning adenovirus genes are present in the tumour cells. It seems that virus genes together with hormonal changes activate the cell protooncogenes giving rise to and maintaining tumours.
Genes required for the full expression of Shigella virulence are on both the chromosome and a large virulence-associated plasmid. Expression of one or more virulence (vir) genes is temperature-regulated, wild-type strains being virulent (invasive) when grown at 37 degrees C but phenotypically avirulent (noninvasive) at 30 degrees C. A vir::lac operon fusion located on the virulence plasmid, which brings the lac genes under control of a temperature-regulated vir gene promoter, was used to select regulatory mutants constitutive for the Lac+ phenotype at the nonpermissive temperature. A transposon Tn10-induced mutant that was Lac+ at 30 degrees C and 37 degrees C was isolated, and the Tn10 insertion was transduced into a wild-type strain. The transductants all simultaneously became deregulated for virulence and invaded HeLa cells equally well at 30 degrees C and 37 degrees C. Other virulence-associated phenotypes were also deregulated and expressed at 30 degrees C. Southern hybridization with a probe for Tn10 determined the insertion to be on the chromosome. Fine mapping by transduction with phage P1L4 positioned the mutation between the galU and trp genes. A cosmid cloned fragment of Shigella chromosomal DNA containing the region around galU was used in complementation studies and showed that the closely linked regulatory gene was able to complement, in trans, the Tn10-induced mutation. We propose that this mutation defines a regulatory gene, virR, and that insertion of Tn10 into this gene inactivates a repressor that normally blocks expression of vir genes at 30 degrees C.
This report describes twin girls with typical features of ataxia-telangiectasia, including increased alpha-fetoprotein, radio-resistant DNA synthesis, characteristic chromosome abnormality, and immunodeficiency. They have, in addition, microcephaly and mental retardation. Complementation studies were performed utilizing Sendai virus--mediated fusion of fibroblast cell lines. Complementation was observed with patients in ataxia-telangiectasia complementation groups A, C, and E but not with the cell line from a patient with the Nijmegen breakage syndrome, in which patients have microcephaly, radio-resistant DNA synthesis, chromosome aberrations, and immunodeficiency but lack ataxia and telangiectasia. These data suggest that the Nijmegen breakage syndrome and the patients described here are not genetically distinct entities but form a spectrum of one disorder.
Complementation studies, using fused cell lines from patients with peroxisomal disorders, have shown correction of defective plasmalogen synthesis and phytanic acid oxidation as well as an increase in the number of peroxisomes. At least six complementation groups have been reported. We demonstrate here that complementing cell lines also acquire the ability to oxidize very long chain fatty acids (VLCFA), and that complementation groups defined with this technique are identical to those reported previously when plasmalogen synthesis was used as the criterion for complementation. This VLCFA complementation technique is of particular value in the study of patients in whom defective VLCFA is the only or major enzymatic defect, and we show complementation between cell lines from two patients each with an isolated defect in one of the peroxisomal fatty acid beta-oxidation enzymes.
In Caulobacter crescentus, mutations have been isolated in more than 30 flagellar genes (fla, flb, and flg) which are required in the cell cycle event of flagellum biogenesis. The flaF and flaG mutations and two newly identified mutations, flbT and flbA (P.V. Schoenlein and B. Ely, J. Bacteriol. 171:000-000, 1989), have been localized to the flaFG region. In this study, the genetic and physical organization of this region was analyzed, using the cloned 4.0-kilobase flaFG region in the recombinant plasmid pPLG727. Plasmid pPLG727 complemented flaF, flaG, flbA, and flbT mutations. Further complementation studies with pPLG727 derivatives indicated that flaF and flbT are unique but overlapping transcription units, whereas flbA and flaG constitute a single transcription unit. To determine the direction of transcription of the putative flbA-flaG operon, the promoterless chloramphenicol transacetylase gene was inserted into various positions in the flbA-flaG region, and merodiploid strains containing these transcriptional fusions were assayed for gene function and expression of chloramphenicol resistance. These studies showed that transcription proceeds from flbA to flaG. To confirm the complementation analysis, Southern analyses were performed on chromosomal DNAs isolated from strains containing insertion and deletion mutations. Taken together, these studies defined the relative gene order at one end of the flaYG flagellar gene cluser as flgL-flaF-flbT-flbA-flaG.
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Cold-sensitive mutations in the secD locus of Escherichia coli result in severe defects in protein export at the non-permissive temperature of 23 degrees C. DNA sequence of a cloned fragment that includes the secD locus reveals open reading frames for seven polypeptide chains. Both deletions and TnphoA insertions in this clone have been used in maxicell and complementation studies to define the secD locus and its products. The secD mutations fall into two complementation groups, defining genes we have named secD and secF. These two genes comprise an operon, the first case of two genes involved in the export process being co-transcribed. The DNA sequence of the two genes along with alkaline phosphatase fusion analysis indicates that they code for integral proteins of the cytoplasmic membrane. We suggest that these two proteins may form a complex in the membrane which acts at late steps in the export process.
In a series of patients with acute pancreatitis we have studied complement factors, antiproteases (alpha 2-macroglobulin and alpha 1-antiprotease) and C-reactive protein to determine the value of their sequential measurement in the prediction of outcome relative to clinical assessment and current multiple factor scoring systems. Complement factors were unhelpful in predicting the severity of an attack. alpha 2-Macroglobulin levels were significantly lower in complicated attacks during days 3-8 and alpha 1-antiprotease levels were significantly higher during days 4-8. C-reactive protein concentrations showed the best discrimination between mild and complicated attacks, levels rising higher and persisting for longer in complicated attacks; these differences were highly significant from day 2 (the morning after admission) to day 8. The concentrations providing the best discrimination were found to be greater than or equal to 210 mg/l for the peak C-reactive protein (on the second, third or fourth day) and greater than or equal to 120 mg/l for the C-reactive protein at the end of the first week. Analysis demonstrated both the peak or seventh-day C-reactive protein concentration to be of similar accuracy to either the Ranson or Glasgow multiple factor scoring systems and slightly better for attacks associated with gallstones. The C-reactive protein assay is simple, quick to perform, provides useful clinical information and is more likely to be of value and to be adopted into routine clinical practice than multiple factor scoring systems.