Mutations In Complementation Group Research Articles

PEX1 mutations in complementation group 1 of Zellweger spectrum patients correlate with severity of disease.

The peroxisome biogenesis disorders (PBD) are a group of autosomal-recessive diseases with complex developmental and metabolic phenotypes, including the Zellweger spectrum and rhizomelic chondrodysplasia punctata. The diseases are caused by defects in peroxisomal matrix protein import and are characterized by the loss of multiple peroxisomal metabolic functions. In humans, 12 complementation groups have been identified, with complementation group 1 accounting for more than two thirds of all PBD patients. Mutations in the PEX1 gene encoding a member of the AAA protein family of ATPases are responsible for the defects in this group, and a variety of PEX1 mutant alleles have been described. We characterized the PEX1 gene mutations and associated haplotypes in a group of thoroughly documented Zellweger spectrum patients in complementation group 1 who represent the broad range of phenotypic variation. We compared the type of mutation with the age of survival, clinical manifestations, and biochemical alterations and found a close relationship between genotype and age of survival. Missense mutations cause a milder form of disease, whereas insertions, deletions, and nonsense mutations are associated with severe clinical phenotypes. Thus, knowing the PEX1 gene mutation is helpful in predicting the course of disease in individual cases.

PEX1 Mutations in Complementation Group 1 of Zellweger Spectrum Patients Correlate with Severity of Disease

PEX1 Mutations in Complementation Group 1 of Zellweger Spectrum Patients Correlate with Severity of Disease

The Active Oxygen Response of Cell Suspensions to Incompatible Bacteria Is Not Sufficient to Cause Hypersensitive Cell Death.

The inoculation of tobacco (Nicotiana tabacum L.) suspension cells with bacterial pathogens that elicit the hypersensitive response (HR) in leaves has been shown to elicit production of active oxygen. This response occurs in two phases, the second of which occurs 1 to 3 h after bacterial addition and is unique to HR-causing interactions. The relationship between the phase II active oxygen response and the HR was characterized using Pseudomonas syringae pv syringae and P. fluorescens (pHIR11), which contains a cosmid clone of the hrp/hrm region from P. syringae pv syringae. TnphoA mutations in complementation groups II through XIII of the hrp cluster blocked the phase II active oxygen response, whereas mutations in the group I hrmA locus did not affect phase II. Despite the normal active oxygen response, bacteria with mutations in the hrmA region did not cause the HR in intact tobacco leaves nor did they induce hypersensitive cell death in cell suspensions. The data indicate that the bacteria do not require the hrmA region to elicit active oxygen production, but a full and intact hrp/hrm region is required to elicit hypersensitive cell death. Therefore, the phase II active oxygen response does not directly cause hypersensitive cell death nor is the response itself sufficient to trigger the HR.

The complete hrp gene cluster of Pseudomonas syringae pv. syringae 61 includes two blocks of genes required for harpinPss secretion that are arranged colinearly with Yersinia ysc homologs.

Pseudomonas syringae pv. syringae 61 contains a 25-kb hrp cluster that is sufficient to elicit the hypersensitive response (HR) in nonhost plants. Previous studies have shown that mutations in complementation groups VIII, IX, and XI in the hrp cluster abolished the ability of the bacterium to cause the HR. The sequence of a 3.7-kb SmaI-SstI fragment covering groups VIII and IX now reveals five open reading frames (ORFs) in the same transcript, designated as hrpU, hrpW, hrpO, hrpX, and hrpY, and predicted to encode proteins of 14,795, 23,211, 9,381, 28,489, and 39,957 Da, respectively. The hrpU, hrpW, hrpO, hrpX, and hrpY genes are homologous and arranged colinearly with the yscQ/spa33/spaO, yscR/spa24/spaP, yscS/spa9/spaQ, yscT/spa29/spaR, and yscU/spa40/spaS genes of Yersinia spp., Shigella flexneri, and Salmonella typhimurium, respectively. These proteins also show similarity to Fli/Flh proteins of Bacillus and enteric bacteria. The Ysc and Spa proteins are involved in the secretion of virulence factors, like the Yop and Ipa proteins. Fli/Flh proteins are involved in flagellar biogenesis. The sequence of a 2.9-kb EcoRV-EcoRI DNA fragment containing mainly group XI revealed five ORFs, designated hrpC, hrpD, hrpE, hrpF, and hrpG, predicted to encode proteins of 29,096, 15,184, 21,525, 7,959, and 13,919 Da, respectively. The first three genes belong to an operon containing hrpZ, which encodes an extracellular protein that elicits the HR. hrpF and hrpG are two potential ORFs upstream of hrpH in the hrpH operon. HrpC is homologous to Yersinia YscJ, Pseudomonas solanacearum HrpI, Xanthomonas compestris pv. vesicatoria HrpB3, and Rhizobium fredii NolT. HrpE is similar to YscL of Yersinia spp. P. s. pv. syringae 61 Hrp proteins are most similar to Ysc proteins among those homologs. TnphoA insertions in hrpC, hrpE, hrpW, hrpX, and hrpY abolished the ability of P. s.pv. syringae 61 to secrete HrpZ (harpinPss), as determined by immunoblot analysis of cell-bound and culture supernatant fractions. Thus, many of the proteins required for flagellar biogenesis and virulence protein secretion in plant and animal pathogens may have a common ancestry.

Mapping of the genes of some components of the electron transport chain (complex I) on the X chromosome of mammals.

This paper describes genetic mapping studies with several respiration-deficient mutants of Chinese hamster fibroblasts which have a defect in complex I of the electron transport chain (NADH-coenzyme Q reductase). The mutations associated with two different complementation groups map on the X chromosome. In two cases (G14 and G20) karyotypic and isozyme analyses in hybrids have shown that a gene(s) on the mouse X chromosome complements the mutation(s) in the hamster cell mutant(s). A cosegregation analysis in hybrid cells has shown the corresponding genes to be linked to the HPRT genes (hamster-mouse hybrids of G14, and hamster-hamster hybrids for G14 and G20). By the same method the defective gene in a third mutant (G4) was also shown to be X-linked. A mutation representing a third complementation group (G11) was shown to be on an autosomal gene. These results provide an explanation for our observation that cells with recessive mutations in complementation groups I and II can be selected at relatively high frequencies.

Assembly of membrane glycoproteins studied by phenotypic mixing between mutants of vesicular stomatitis virus and retroviruses

Temperature sensitive ( ts) mutations of vesicular stomatitis virus (VSV), Indiana serotype, which belong to complementation group V ( tsV) have been shown to affect the viral envelope glycoprotein, or G protein. When ts V mutants are grown in cells producing avian leukosis viruses, the titers of infectious VSV obtained at the nonpermissive temperature are 10 4-fold higher than in control cells. Cells releasing murine leukemia viruses or avian reticuloendotheliosis virus rescue VSV ts V mutants much less efficiently. The rescued virions have the properties of envelope pseudotypes in that their host range is restricted to that of the helper retrovirus, they are neutralized by anti-retrovirus antibodies but not anti-VSV antibodies, and they are not thermolabile. Sensitive serological techniques, including the use of complement-mediated virolysis, immunoprecipitation, and monoclonal antibody reacting with G protein, show that VSV pseudotypes produced at the nonpermissive temperature have no detectable G protein, whereas VSV particles released from retrovirus infected cells at the permissive temperature have mosaic envelopes bearing both VSV G protein and retrovirus glycoprotein. In mixed infections of Rous sarcoma virus (RSV) and VSV ts V mutants, pseudotype particles with RSV genomes and VSV envelope antigens are produced only at the permissive temperature. In contrast, substantial yields of RSV(VSV) pseudotypes but no VSV(RSV) pesudotypes are obtained at the nonpermissive temperature with VSV carrying mutations in complementation group III, which affect M protein. Thermolabile VSV tsV mutants form RSV(VSV) pseudotypes which also are thermolabile. The kinetics of heat inactivation of G protein function in tsV mutants is the same in VSV particles with unmixed envelopes and with mosaic envelopes. From these studies of phenotypic mixing we draw the following conclusions: (i) The synthesis of functional M protein but not G protein is essential for the maturation of VSV virions. (ii) VSV M protein is not required for the assembly of G protein into retrovirus virions. (iii) The thermolabile nature of tsV VSV mutants is an intrinsic property of the G protein, independent of the type of virion into which it is incorporated and of other viral glycoproteins which may be assembled into the envelope of the same virion.

Envelope proteins of vesicular stomatitis virus: effect of temperature-sensitive mutations in complementation groups III and V.

All five major viral proteins were synthesized in chicken embryo cells infected with vesicular stomatitis virus temperature-sensitive (ts) mutants of complementation groups III and V and maintained at the nonpermissive temperature. The distribution of these proteins among cytoplasmic cellular fractions separated on discontinuous sucrose gradients was identical for wild-type and tsIII-infected cells. Strikingly different patterns were observed for the G protein in gradients from cells infected by tsV mutants; very little, if any, G protein was found in the lightest fraction. Pulse and chase experiments with wild-type, virus-infected cells showed that protein G moves from the heaviest to the lightest fraction before being incorporated into the virion. After shift down to the permissive temperature (30 C), G protein synthesized at 39.6 C in tsV-infected cells became associated with the lightest cellular fraction and later with the released virions. In contrast, M protein, synthesized at 39.6 C in tsIII-infected cells, was not incorporated into the virions after shift down. These data strongly suggest, first, that M protein is encoded by the vesicular stomatitis gene III, and second, that incorporation of G protein in the lightest cellular fraction is a necessary step of vesicular stomatitis maturation. This step is impaired by tsV mutations.

Transcription and replication of vesicular stomatitis virus: effects of temperature-sensitive mutations in complementation group IV.

Temperature-sensitive (ts) mutants of vesicular stomatitis virus belonging to the RNA(-) complementation group IV were investigated under various conditions to study both their RNA and protein syntheses. In infected cells maintained at 39.2 C, viral RNA species were recovered only in the 13 to 15S region of the gradient in an amount depending on the ts mutant used. In the presence of cycloheximide at 39.2 C, the primary transcription was deficient, especially for 28S mRNA production. When mutant-infected cells were shifted to nonpermissive temperature, a shutoff of 28S mRNA synthesis occurred as a general feature. On the contrary under this condition, the two mutants chosen, ts IV100 and ts IV111, behaved very differently in their 13 to 15S and 38S RNA production. However, treatment with cycloheximide at the time of the transfer to 39.2 C resulted in a similar recovery of 13 to 15S RNA in both mutants, whereas the 28S remained very depressed. The viral proteins synthesized by cells infected with the same two mutants also showed a distinct pattern, especially regarding the N protein; a correlation between 38S RNA and protein N syntheses was tentatively drawn. The whole set of data suggested that the lesion in group IV mutants concerned a viral structural protein required for the process of in vivo transcription and which probably intervened in the replication mechanism.