An ensemble method for identifying regulatory circuits with special reference to the qa gene cluster of Neurospora crassa.

A chemical reaction network for the regulation of the quinic acid (qa) gene cluster of Neurospora crassa is proposed. An efficient Monte Carlo method for walking through the parameter space of possible chemical reaction networks is developed to identify an ensemble of deterministic kinetics models with rate constants consistent with RNA and protein profiling data. This method was successful in identifying a model ensemble fitting available RNA profiling data on the qa gene cluster.

Radioactivity-based and spectrophotometric assays for isoorotate decarboxylase: identification of the thymidine salvage pathway in lower eukaryotes.

A few organisms, notably some fungi, have the ability to metabolize thymidine to uracil, thus conserving the pyrimidine ring for subsequent metabolic use. Neurospora crassa possesses this pathway, termed the thymidine salvage pathway, and can utilize thymidine as a total pyrimidine source. The enzyme isoorotate decarboxylase (IDCase) completes this pathway via the enzymatic removal of the carboxylate from isoorotate to yield uracil. We describe in this communication two assays for IDCase and their application to determine activity levels, kinetic constants, and inhibitory properties. One uses [carboxy-14C]isoorotate from which the enzymatically generated 14CO2 is collected and quantitated. The second assay utilizes the spectral difference between 2-thioisoorotate and its decarboxylated product, 2-thiouracil. The spectral difference is greatest at 334 nm, out of the range of absorbance of total protein and thus usable for a spectrophotometric assay. The assays are sufficiently sensitive and accurate to be used in the measurement of Km values for both substrates. IDCase activity is found to be significantly higher in N. crassa strains lacking uc-1, a putative regulatory gene, suggesting a degree of metabolic control over this pathway. 5-Nitrouracil is found to inhibit IDCase with an estimated Ki value that is too low for accurate determination.

Chromosome rearrangements recovered following transformation of Neurospora crassa.

New chromosome rearrangements were found in 10% or more of mitotically stable transformants. This was shown for transformations involving a variety of different markers, vectors and recipient strains. Breakpoints were randomly distributed among the seven linkage groups. Controls using untransformed protoplasts of the same strains contained almost no rearrangements. A study of molecularly characterized Am+ transformants showed that rearrangements are frequent when multiple ectopic integration events have occurred. In contrast, rearrangements are absent or infrequent when only the resident locus is restored to am+ by a homologous event. Sequences of the transforming vector were genetically linked to breakpoints in 6 of 10 translocations that were examined using Southern hybridization or colony blots.

Use of gene replacement transformation to elucidate gene function in the qa gene cluster of Neurospora crassa.

Gene replacement by transformation, employing selective genetic recombination techniques, has been used to delete or disrupt the qa-x, qa-y and qa-1S genes of the qa gene cluster of Neurospora crassa. The growth characteristics of the strain carrying the deletion of the qa-y gene support earlier evidence that this gene encodes a quinic acid permease. The strain containing the deletion of the qa-1S gene (delta qa-1S) was examined with respect to quinic acid induction and carbon catabolite repression. The delta qa-1S strain exhibits constitutive expression of the qa genes supporting earlier evidence that the qa-1S gene codes for a repressor. Several of the qa genes continued to be expressed at high levels even in the presence of glucose in the delta qa-1S strain, which indicates that transcription of these genes is not being affected directly by a repressor molecule in the presence of glucose.

Analysis of junction sequences resulting from integration at nonhomologous loci in Neurospora crassa.

We have analyzed the junctions involved in two examples of ectopic integration of plasmids containing the am+ (glutamate dehydrogenase) gene into a strain of Neurospora crassa bearing a complete deletion of the am locus. In one transformed strain a single copy of plasmid DNA had been integrated into linkage group (LG) III DNA without the loss of chromosomal DNA. In contrast, 450 bp had been lost from plasmid sequences at the site of integration. The transforming DNA used was circular, so we postulate that the plasmid was linearized and truncated prior to its integration by end joining into a break in LG III DNA. There was no significant homology between the incoming DNA and DNA at the site of integration. The second transformed strain resulted from transformation with a linearized plasmid. It contained multiple integrated copies of plasmid DNA, one of which was recloned, together with adjacent chromosomal DNA, by plasmid rescue in Escherichia coli. Prior to integration into chromosomal DNA, the linear plasmid had been truncated by 64 bp on one end and 3.2 kbp on the other end. One end of the integrated DNA was adjacent to DNA from the right arm of LG I, while the other end was integrated into a copy of a repetitive sequence. Restriction fragment length polymerism mapping showed that integration was in a copy of the repetitive sequence that is linked to the previously unassigned telomere M11 and is distantly linked to the LG VI marker con-11. Genetic analysis revealed that a long segment of LG I containing all markers from un-1 to the right tip has been translocated to the right end of LG VI. Tetrad analysis showed that the integrated DNA was closely linked to the translocation. We conclude that the transforming DNA was truncated and joined to DNA from two different chromosomes by end joining during the formation of a quasiterminal translocation, T(IR----VIR) UK-T12. We also conclude that the previously unassigned telomere, M11, is the right end of LG VI.

Sequence and characterization of the met-7 gene of Neurospora crassa.

The proteins encoded by the met-7+ and met-3+ genes of Neurospora crassa are required to form a functional cystathionine-gamma-synthase (CGS). The met-7+ gene has been cloned by complementation of a met-7 mutant. The nucleotide sequence of the complementing DNA reveals the presence of a 542-amino acid open reading frame (ORF). Disruption of this ORF abolishes complementation of the met-7 mutation.

Comparative studies of the quinic acid (qa) cluster in several Neurospora species with special emphasis on the qa-x-qa-2 intergenic region.

The organization of the quinic acid (qa) genes in Neurospora crassa has been compared to that in several other Neurospora species. This gene cluster was found to be highly conserved in all species examined. However, there are numberous restriction fragment length polymorphisms that distinguish the heterothallic and homothallic species. Catabolic dehydroquinase assays indicated that qa-2 gene expression in the homothallic species is subject to induction by quinic acid, as is the case in N. crassa. The qa-x-qa-2 intergenic region of the homothallic species N. africana was cloned and sequenced. Conserved qa activator (qa-1F) binding sites have been identified in this region. When the qa-x-qa-2 intergenic region of N. crassa was replaced with its N. africana counterpart, qa-2 gene expression was reduced; however repression by glucose appeared normal. Furthermore, the N. africana start site for qa-2 transcription (which differs from the N. crassa start site) was utilized in the transformant. The overall evidence suggests that a weakening of the -120 activator binding site in the qa-x-qa-2 intergenic region may be responsible for these differences.

The Wilhelmine E. Key 1989 invitational lecture. Organization and regulation of the qa (quinic acid) genes in Neurospora crassa and other fungi.

In Neurospora crassa, five structural genes and two regulatory genes control the use of quinic acid as a carbon source. All seven genes are tightly linked to form the qa gene cluster. The entire cluster, which has been cloned and sequenced, occupies a continuous DNA segment of 17.3 kb. Three pairs of genes are divergently transcribed, including the two regulatory genes that are located at one end of the cluster and that encode an activator (qa-1F) and a repressor (qa-1S). Three of the structural genes (qa-2, qa-3, and qa-4) encode inducible enzymes that catalyze the catabolism of quinic acid. One structural gene (qa-y) encodes a quinate permease; the function of the fifth gene (qa-x) is still unclear. Present genetic and molecular evidence indicates that the qa activator and repressor proteins and the inducer quinic acid interact to control expression at the transcriptional level of all the qa genes. The activator, the product of the autoregulated qa-1F gene, binds to symmetrical 16 base pair upstream activating sequences located one or more times 5' to each of the qa genes. A conserved 28 amino acid sequence containing a six cysteine zinc binding motif located in the amino terminal region of the activator has been directly implicated in DNA binding. Evidence for other functional domains in the activator and repressor proteins are discussed. Indirect evidence suggests that the repressor is not a DNA-binding protein but forms an inactive complex with the activator in the absence of the inducer.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship of vector insert size to homologous integration during transformation of Neurospora crassa with the cloned am (GDH) gene.

We used lambda and plasmid vectors containing the am+ gene in an insert of from 2.7 to 9.1 kb, to transform am point mutant and deletion strains. A total of 199 transformants were examined with the potential to yield am+ transformants by homologous recombination. When we used vectors that had 9.1 kb of homology with the chromosomal DNA, 30% of the transformants obtained were the result of homologous recombination regardless of whether the vector was a lambda molecule, a circular plasmid, or a plasmid that had been linearized prior to transformation. When vectors with up to 5.1 kb of homology were used, very few transformants (1 of 89 tested) resulted from homologous recombination. Of a sample of 29 ectopic integration events obtained by transformation with the 9.1 kb fragment cloned in a lambda vector, 18 included a major part (usually almost all) of both arms of lambda with the entire Neurospora 9.1 kb insert between them. Four included only lambda long arm sequence together with an adjacent segment of the insert containing the am gene. The remaining seven were the result of multiple integrations. There was no evidence of circularization of the lambda vector prior to integration. All transformants that had multiple copies of the am gene appeared to be subject to the RIP process, which causes multiple mutations in duplicated sequences during the sexual cycle.

Use of transformation to make targeted sequence alterations at the am (GDH) locus of Neurospora.

Specific in vitro-generated insertion, replacement, and deletion mutations have been integrated near the chromosomal locus of am (NADP-specific glutamate dehydrogenase) of Neurospora crassa. Two approaches have been successful. One approach used am+-containing vectors capable of integrating at any site in the genome. This technique was used to introduce a specific 700 bp insertion near the am locus and to replace chromosomal sequences near am with plasmid DNA. Efficiency was low, however, and many transformants had to be screened to find the desired alterations among the ectopic insertions unless the incoming DNA had a large region of homology with the am region. A second approach increased the efficiency by using vectors containing a truncated am gene, so that prototrophs could arise only by homologous recombination. Overall transformation frequency was reduced relative to the first method, but a large fraction of the transformations involved specific alterations of the am region.

Isolation and preliminary characterization of a plasmid mutant derepressed for conjugal transfer in Staphylococcus aureus.

The plasmid pCRG1600 is a 52.9-kb self-transmissible plasmid coding for resistance to aminoglycoside and beta-lactam antibiotics in Staphylococcus aureus. When transferred by transduction, plasmid deletion mutants affecting one or more antibiotic-resistance genes were readily obtained. Of these, one derivative (pCRG1690) was found to exhibit a conjugal transfer frequency ca. 100-fold higher than that of the wild-type plasmid. A preliminary physical-genetic map of pCRG1600 located tra in a 14.6-kb region within the 16.9-kb XbaI-A fragment. An 8.5-kb deletion to the left of tra in pCRG1690 was specifically associated with the increased conjugal transferability of the plasmid. Thus, pCRG1690 appears similar to plasmids derepressed for conjugal transfer (drd) in gram-negative bacterial species.