[When genetics matters: an unusual case of left-sided hemiparesis]. / Wenn der Stammbaum zählt: ein ungewöhnlicher Fall einer linksseitigen Hemiparese.

We report the case of a 35-year-old woman who has been admitted to our emergency room because of the sudden onset of a left-sided hemiparesis. The physical examination showed disseminated teleangiectases on the upper and lower lip, on the mucosal surface of the tongue and on the skin. The cerebral CT scan presented a right-sided fronto-parietal lesion, more likely an abscess, confirmed on surgical removal. A meticulous family history showed the presence of similar clinical features among several family members, raising the certainty about the presence of the Rendu-Osler-Weber disease. This case is highly instructive in emphasizing the value of taking a careful medical history and how doing it is extremely important in our daily practice.

Mutants of the carotene cyclase domain of al-2 from Neurospora crassa.

Phytoene synthase and carotene cyclase, two key enzymes in carotenoid biosynthesis, are encoded by two separate genes in bacteria and plants, but by a single bifunctional gene in fungi. The cyclase function has been demonstrated for the products of the genes crtYB from the basidiomycete Xanthophyllomyces dendrorhous, and for carRA and carRP from the zygomycetes Phycomyces blakesleeanus and Mucor circinelloides, respectively. These three genes are highly similar to al-2 from Neurospora crassa. Taking advantage of the high proportion of the final product of the carotenoid pathway that accumulates Neurospora when mycelium is illuminated at low temperature, we have isolated two mutants with a pale reddish pigmentation. Both mutants are complemented by the wild-type al-2 gene, and carry mutations in the al-2 domain to which cyclase activity has been attributed in other fungi. The mutants lack neurosporaxanthin and accumulate an unidentified reddish carotenoid, as shown by column chromatography and HPLC. The chemical and spectrophotometrical properties of this carotenoid are consistent with the absence of carotenoid cyclization, and indicate that the product of al-2 is bifunctional. The existence of a single gene responsible for phytoene synthase and carotene cyclase thus seems to be a widespread trait among filamentous fungi, as shown by the examples now known in a basidiomycete, two zygomycetes and one ascomycete.

Genome analysis in Neurospora crassa; cloning of four loci arginine-1 (arg-1), methionine-6 (met-6), unknown-7 (un-7), and ribosome production-1 (rip-1) and associated chromosome walking.

We have cloned four Neurospora crassa genes by complementation analysis. Cloned genes include the arginine-1 (arg-1), methionine-6 (met-6), unknown-7 (un-7), and ribosome production-1 (rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of the Neurospora genome is covered in these walks.

Genome analysis on linkage group VI of Neurospora crassa.

Two chromosome walks covering 420 and 110 kb on the left arm of linkage group VI (LGVIL) of Neurospora crassa were purscrooued with the goal of cloning carotenogenic loci. Complementation analysis with clones isolated in the 420-kb walk allowed identification of the yellow-1 (ylo-1) gene which is essential for Neurospora carotenogenesis. We have physically located a second gene, unknown-13 (un-13), between the cross-pathway control-1, (cpc-1) and ylo-1 loci. Cloning of a second potential carotenogenic locus, vivid (vvd), from our walks was attempted using screening of Northern blots with radiolabeled DNA fragments from walk clones to identify gene transcripts. The radiolabeled DNA fragments were used to clone complementary DNA isolates representing an additional four genes in the two walks.

Developmental and photoregulation of three Neurospora crassa carotenogenic genes during conidiation induced by desiccation.

Neurospora crassa asexual sporulation (conidiation) is induced by different cues including desiccating aerial environments. Three of the genes that are expressed during this developmental pathway are the albino (al) genes, which encode carotenoid biosynthetic enzymes. If conidiation is induced by nutrient deprivation in liquid culture, two overlapping al-3 mRNAs, al-3 (m) and al-3 (c), are expressed (Arpaia et al., 1995, Dev. Biol. 170, 620-635). Here we quantitate accumulation of each of the four albino gene messages, al-1, al-2, al-3 (m), and al-3 (c), in conidiating wild-type cultures and in cultures of two mutants defective in photoinduced carotenogenesis, wc-1 and wc-2. Of the four albino gene transcripts, only al-1 expression is developmentally regulated in wild-type cultures conidiating in darkness. Expression of all four albino gene transcripts is developmentally regulated in conidiating wc-1 or wc-2 cultures. Unlike other albino gene transcripts, expression of al-3 (c) is photoregulated only early in conidiation. The fld and fl mutants are defective in conidiation with development halting at distinct early stages of spore formation. In desiccated fld and fl cultures al-1, al-2, and al-3 (m) transcript accumulation is observed to peak early and then decline, while al-3 (c) transcript accumulation increases with time.

Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation.

The molecular mechanisms involved in transgene-induced gene silencing ('quelling') in Neurospora crassa were investigated using the carotenoid biosynthetic gene albino-1 (al-1) as a visual marker. Deletion derivatives of the al-1 gene showed that a transgene must contain at least approximately 132 bp of sequences homologous to the transcribed region of the native gene in order to induce quelling. Transgenes containing only al-1 promoter sequences do not cause quelling. Specific sequences are not required for gene silencing, as different regions of the al-1 gene produced quelling. A mutant defective in cytosine methylation (dim-2) exhibited normal frequencies and degrees of silencing, indicating that cytosine methylation is not responsible for quelling, despite the fact that methylation of transgene sequences frequently is correlated with silencing. Silencing was shown to be a dominant trait, operative in heterokaryotic strains containing a mixture of transgenic and non-transgenic nuclei. This result indicates that a diffusable, trans-acting molecule is involved in quelling. A transgene-derived, sense RNA was detected in quelled strains and was found to be absent in their revertants. These data are consistent with a model in which an RNA-DNA or RNA-RNA interaction is involved in transgene-induced gene silencing in Neurospora.

Developmental and photoregulation of al-1 and al-2, structural genes for two enzymes essential for carotenoid biosynthesis in Neurospora.

The levels of al-1 and al-2 transcripts change dramatically in response to light and development during the formation of Neurospora crassa asexual spores (conidia). al-1 and al-2 mRNAs accumulate throughout conidiation irrespective of lighting conditions initially at low levels. As conidiation proceeds, two increases in albino message accumulation are observed. This developmentally induced photoindependent message accumulation was not observed in Neurospora mutants blocked at relevant stages of conidiation. During conidiation, light induces additional accumulation of al-1 and al-2 gene-specific transcripts; the photoinduced increase in albino gene transcript levels was not observed in two Neurospora mutants, wc-1 and wc-2, that are defective in all physiological photoresponses.

Characterization of al-2, the phytoene synthase gene of Neurospora crassa. Cloning, sequence analysis, and photoregulation.

We have cloned the al-2 gene of Neurospora crassa and have analyzed its structure and regulation. The gene encodes a 603-residue polypeptide with a segment homologous to prokaryotic and other eukaryotic phytoene synthases. RNA measurements showed that the level of al-2 mRNA increased over 30-fold in photoinduced mycelia compared with dark-grown mycelia. This observation is consistent with the fact that carotenoid biosynthesis is induced by blue light during growth of N. crassa mycelia. The photoinduced increase in al-2 mRNA levels was not observed in two Neurospora mutants, wc-1 and wc-2, that are defective in all physiological photoresponses.

Cloning, sequence, and photoregulation of al-1, a carotenoid biosynthetic gene of Neurospora crassa.

Carotenoid biosynthesis is regulated by blue light during growth of Neurospora crassa mycelia. We have cloned the al-1 gene of N. crassa encoding the carotenoid-biosynthetic enzyme phytoene dehydrogenase and present an analysis of its structure and regulation. The gene encodes a 595-residue polypeptide that shows homology to two procaryotic carotenoid dehydrogenases. RNA measurements showed that the level of al-1 mRNA increased over 70-fold in photoinduced mycelia. Transcription run-on studies indicated that the al-1 gene was regulated at the level of initiation of transcription in response to photoinduction. The photoinduced increase of al-1 mRNA levels was not observed in two Neurospora mutants defective in all physiological photoresponses. Analysis of cosmid containing al-1 and of a translocation strain with a breakpoint within al-1 indicated that al-1 transcription proceeds towards the centromere of linkage group I of N. crassa.

Carotenoid desaturases from Rhodobacter capsulatus and Neurospora crassa are structurally and functionally conserved and contain domains homologous to flavoprotein disulfide oxidoreductases.

The characteristic red color of some photosynthetic bacteria and the orange color of Neurospora conidia is due to the presence of carotenoids, photoprotective pigments synthesized by plants, algae, bacteria, and fungi. Generally, carotenoids are tetraterpenes in which absorption of visible light and photoprotection are mediated by a chain of conjugated double bonds, the chromophore, which is formed by successive desaturations of phytoene, a colorless precursor. The genes al-1 and crtI mediate the desaturation of phytoene in Neurospora crassa and Rhodobacter capsulatus, respectively. Here, we report that alignment of the primary sequence of Al-1, CrtI, and CrtD, another carotenoid desaturase, reveals conservation with amino acid residues that mediate FAD-binding and dimerization functions in Azotobacter vinelandii dihydrolipoamide dehydrogenase and human glutathione reductase, two disulfide oxidoreductases. Plasmids containing the coding region of an al-1 cDNA fused to appropriate bacterial transcriptional and translational signals complement crtI mutants. Our results indicate that both structure and function of carotenoid desaturases have been conserved during evolution and suggest that these enzymes are evolutionarily related to disulfide oxidoreductases.

Host-specific effects of the korA-korB operon and oriT region on the maintenance of miniplasmid derivatives of broad host-range plasmid RK2.

Two genetic determinants are sufficient for small derivatives of broad host-range plasmid RK2 to replicate in different Gram-negative bacteria: trfA, which encodes a replication initiator, and oriV, the origin of replication. In this study, nonessential RK2 determinants in the region encoding oriT, the origin of conjugative transfer, and the korA-korB operon, whose products regulate trfA expression, were tested for their effects on the stability of mini-RK2 plasmids in eight different hosts. We found that determinants of both regions can substantially alter plasmid stability, but the effects are not uniform in all hosts. The results also indicate that the effects of the korA-korB operon extend beyond that of the regulation of trfA transcription. This study further illustrates the different requirements for stable plasmid maintenance in diverse bacteria and the ability of wild-type RK2 to adapt to a variety of intracellular environments. The data also provide further evidence for the involvement of different regions of RK2 for stable maintenance in various hosts.

Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria.

The replication and maintenance properties of the broad-host-range plasmid RK2 and its derivatives were examined in nine gram-negative bacterial species. Two regions of RK2, the origin of replication (oriV) and a segment that encodes for a replication protein (trfA delta kilD, designated trfA*), are sufficient for replication in all nine species tested. However, stable maintenance of this minimal replicon (less than 0.3% loss per generation under nonselection conditions) is observed only in Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, and Azotobacter vinelandii. Maintenance of this minimal replicon is unstable in Rhizobium meliloti, Agrobacterium tumefaciens, Caulobacter crescentus, Acinetobacter calcoaceticus, and Rhodopseudomonas sphaeroides. A maintenance function has been localized to a 3.1-kilobase (kb) region of RK2 encoding three previously described functions: korA (trfB korB1 korD), incP1-(II), and korB. The 3.1-kb maintenance region can increase or decrease the stability of maintenance of RK2 derivatives dependent on the host species and the presence or absence of the RK2 origin of conjugal transfer (oriT). In the case of A. calcoaceticus, stable maintenance requires an RK2 segment that includes the promoter and the kilD (kilB1) functions of the trfA operon in addition to the 3.1-kb maintenance region. The broad-host-range maintenance requirements of plasmid RK2, therefore, are encoded by multiple functions, and the requirement for one or more of these functions varies among gram-negative bacterial species.

Comparison of 10 IncP plasmids: homology in the regions involved in plasmid replication.

We have examined the DNA homology in the replication regions of 10 IncP plasmids independently isolated from several different countries. Two regions of RK2, the best-studied plasmid of this group, are required for vegetative DNA replication: the origin of replication (oriV) and the trfA region, which codes for a gene product necessary for replication. Six of nine IncP plasmids studied were identical to RK2 in the oriV and trfA regions as shown by Southern hybridization. Three P plasmids, R751, R772, and R906, showed weaker homology with the RK2 trfA, region and hybridized to different-sized HaeII fragments than the other six plasmids. R751, R772, and R906 hybridized to the region of the RK2 replication origin which expresses P incompatibility but differed markedly from RK2 and the other six plasmids in the GC-rich region of the origin required for replication. These data indicate that the P-group plasmids can be divided into two subgroups: IncP alpha, which includes the RK2-like plasmids, and IncP beta which includes the R751-like plasmids.

Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression.

Derivatives of plasmid pRK290 that are useful for cloning and for analyzing gene expression in a wide variety of Gram-negative bacteria are described. A smaller broad host range plasmid derived from RK2, with properties similar to that of pRK290, is also described.

Replication of derivatives of the broad host range plasmid RK2 in two distantly related bacteria.

A 0.7-kb segment of the broad host range plasmid RK2 containing the replication origin of this plasmid will replicate in Escherichia coli and Pseudomonas putida when this segment is joined to a 1.8-kb region of RK2 designated traA*. The presence of another region of RK2, designated trfB, that previously was implicated in RK2 replication had no effect on the maintenance of the RK2 trfA*-oriV replicon in these two organisms. These observations indicate a requirement for a minimal account of information for replication of this broad host range plasmid in two distantly related bacteria.