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.

Position paper on fatal abusive head injuries in infants and young children.

This article represents the work of the National Association of Medical Examiners Ad Hoc Committee on shaken baby syndrome. Abusive head injuries include injuries caused by shaking as well as impact to the head, either by directly striking the head or by causing the head to strike another object or surface. Because of anatomic and developmental differences in the brain and skull of the young child, the mechanisms and types of injuries that affect the head differ from those that affect the older child or adult. The mechanism of injury produced by inflicted head injuries in these children is most often rotational movement of the brain within the cranial cavity. Rotational movement of the brain damages the nervous system by creating shearing forces, which cause diffuse axonal injury with disruption of axons and tearing of bridging veins, which causes subdural and subarachnoid hemorrhages, and is very commonly associated with retinal schisis and hemorrhages. Recognition of this mechanism of injury may be helpful in severe acute rotational brain injuries because it facilitates understanding of such clinical features as the decrease in the level of consciousness and respiratory distress seen in these injured children. The pathologic findings of subdural hemorrhage, subarachnoid hemorrhage, and retinal hemorrhages are offered as "markers" to assist in the recognition of the presence of shearing brain injury in young children.

The Neurospora crassa genome: cosmid libraries sorted by chromosome.

A Neurospora crassa cosmid library of 12,000 clones (at least nine genome equivalents) has been created using an improved cosmid vector pLorist6Xh, which contains a bacteriophage lambda origin of replication for low-copy-number replication in bacteria and the hygromycin phosphotransferase marker for direct selection in fungi. The electrophoretic karyotype of the seven chromosomes comprising the 42.9-Mb N. crassa genome was resolved using two translocation strains. Using gel-purified chromosomal DNAs as probes against the new cosmid library and the commonly used medium-copy-number pMOcosX N. crassa cosmid library in two independent screenings, the cosmids were assigned to chromosomes. Assignments of cosmids to linkage groups on the basis of the genetic map vs. the electrophoretic karyotype are 93 +/- 3% concordant. The size of each chromosome-specific subcollection of cosmids was found to be linearly proportional to the size of the particular chromosome. Sequencing of an entire cosmid containing the qa gene cluster indicated a gene density of 1 gene per 4 kbp; by extrapolation, 11,000 genes would be expected to be present in the N. crassa genome. By hybridizing 79 nonoverlapping cosmids with an average insert size of 34 kbp against cDNA arrays, the density of previously characterized expressed sequence tags (ESTs) was found to be slightly <1 per cosmid (i.e., 1 per 40 kbp), and most cosmids, on average, contained an identified N. crassa gene sequence as a starting point for gene identification.

Sexual asphyxiation: an unusual case involving four male adolescents.

A case of sexual asphyxiation is described involving children, adolescent males, homosexuality and two bizarre strangulations. Two adolescent males (ages 13 & 10) initially engaged in homosexual activity in conjunction with sexual asphyxiation. This unusual activity was by chance observed by one of the boy's younger brother and his friend. The younger children (aged 7 & 8) expressed a desire to join in the activity, which they did by letting ligatures be tied around their necks and engaging in anal intercourse to the point of fatal asphyxiation. The case is of interest in the reported way the younger boys discovered the practice (a sexually explicit magazine) and is unusual because of the young ages of the boys involved.

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.

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)

Induced expression of the Aspergillus nidulans QUTE gene introduced by transformation into Neurospora crassa.

The qa-2 gene of Neurospora crassa encodes catabolic dehydroquinase which catabolizes dehydroquinic acid to dehydroshikimic acid. The QUTE gene of Aspergillus nidulans corresponds to the qa-2 gene of N. crassa. The plasmid pEH1 containing the QUTE gene from A. nidulans was used to transform a qa-2- strain of N. crassa. In Southern blot analyses, DNAs isolated from these transformants hybridized specifically to the QUTE gene probe. Northern blot analyses indicated that QUTE mRNA was produced in the transformants. The functional integrity of the QUTE gene in N. crassa was indicated by transformants which had regained the ability to grow on quinic acid as sole carbon source. Enzyme assays indicated that the specific activities of catabolic dehydroquinase induced by quinic acid in the transformants ranged from 4% to 32% of that induced in wild-type N. crassa. The evidence that the QUTE structural gene of A. nidulans is inducible when introduced into the N. crassa genome implies that the N. crassa qa activator protein can recognize, at least to a limited extent, DNA binding sequences 5' to the QUTE gene.

Bialaphos resistance as a dominant selectable marker in Neurospora crassa.

Conidia of Neurospora crassa are sensitive to the herbicide bialaphos at concentrations of 160 mg/l in Westergaard's or Fries' minimal media. Plasmid pJA4 was constructed by inserting a truncated bar gene from Streptomyces hygroscopicus fused to the his-3 promoter from N. crassa into pUC19. The bar gene in plasmid pJA4 confers resistance to bialaphos when transformants are selected on a medium containing bialaphos. The bar gene can be used as an additional dominant selectable marker for transformation of fungi.

DNA sequence, organization and regulation of the qa gene cluster of Neurospora crassa.

In Neurospora, five structural and two regulatory genes mediate the initial events in quinate/shikimate metabolism as a carbon source. These genes are clustered in an 18 x 10(3) base-pair region as a contiguous array. The qa genes are induced by quinic acid and are coordinately controlled at the transcriptional level by the positive and negative regulators, qa-1F and qa-1S, respectively. The DNA sequence of the entire qa gene cluster has been determined and transcripts for each gene have been mapped. The qa genes are transcribed in divergent pairs and two types of transcripts are associated with each gene: basal level transcripts that initiate mainly from upstream regions and are independent of qa regulatory gene control, and inducible transcripts that initiate downstream from basal transcripts and are dependent on qa-1F binding to a 16 base-pair sequence. We discuss how both types of transcription relate to the organization of the qa genes as a cluster and how this may impose constraints on gene dispersal.

Genetical and molecular analyses of qa-2 transformants in Neurospora crassa.

Neurospora crassa qa-2+ transformants from five different donor DNA clones were analyzed by genetical and molecular techniques. None of the 32 transformants have the qa-2+ DNA replacing the qa-2- gene in linkage group VII. In one transformant, the qa-2+ DNA was inserted adjacent to the qa-2- gene. Thirty-one transformants have the qa-2+ inserts at sites not linked, or not closely linked, to the qa-2 locus in LG VII. Plasmid sequences were integrated along with the qa-2+ gene in 28 transformants. In the unlinked duplication-type transformants, catabolic dehydroquinase (the qa-2+ gene product) was induced at 5-100% of the wild-type-induced enzyme activity, with 24 transformants in the 5-80% range. The reduced levels of enzyme activity may be due to "position effects" of sequences adjacent to the integration site either in the N. crassa genomic DNA or in the flanking plasmid (pBR322 or pBR325) sequences. Unexpected gene conversion-like events, in which a qa-2+ gene was changed to qa-2-, were observed in tetrads from intercrosses between unlinked duplication-type transformants and in selfings of such transformants.

Rearrangement mutations on the 5' side of the qa-2 gene of Neurospora implicate two regions of qa-1F activator-protein interaction.

Transcriptional activation of the Neurospora crassa qa genes normally requires the positive regulatory gene, qa-1F+, whose function is controlled by the inducer quinic acid and by the product of the negative regulatory gene, qa-1S+. The properties of qa-1F+ activator have been examined in transcriptional mutations of the qa-2 structural gene, in which activator-independent transcription of qa-2 (qa-2ai mutants) occurs in strains having a qa-1F- gene. Seven qa-2ai mutants with DNA rearrangements in different 5' regions of qa-2 were analyzed in qa-1F+ strains. In five with rearrangements at position -190 or further upstream, expression of the qa-2 gene was inducible, and induction was accompanied by a change in the initiation site for transcription from position -45, characteristic of constitutive initiation in qa-2ai mutants to position +1, characteristic of the induced wild type. In two mutants with breakpoints at positions -86 and -53, qa-2 transcription initiated from upstream sequences within the rearrangements but not at the +1 site, and qa-2 expression was noninducible. The results indicate that (i) sequences between positions -190 and -86 are required for positive control of initiation at position +1, and (ii) negative control does not require sequences downstream of position -86. Additional evidence suggests that the product of the qa-1F+ gene in the noninduced state may also interact with distal upstream sequences positioned midway between divergently transcribed qa genes.

Xylazine in human tissue and fluids in a case of fatal drug abuse.

A fatal case of multiple drug abuse in a 36-year-old veterinarian involving injection of xylazine and ingestion of alcohol and clorazepate is presented. Quantitative analysis of xylazine was by gas liquid chromatography with a nitrogen detector. Xylazine concentrations (mg/L or mg/kg) were: blood, 0.2; brain, 0.4; kidney, 0.6; liver, 0.9; lung, 1.1; omentum adipose 0.05; and urine, 7.0. Blood ethanol and nordiazepam concentrations were 380 mg/dL and 2.5 mg/L, respectively.

Homicide by intravenous injection of naphtha.

A case of homicide by the intravenous injection of Energine, a petroleum distillate spot remover, is presented. This case is the only known homicide committed with naphtha. This elderly man had severe natural disease in addition to chest trauma sustained in the assault leading to death; however, the rapid injection of approximately 25 mL of Energine was the overwhelming cause of death.

Cis-acting and trans-acting regulatory mutations define two types of promoters controlled by the qa-1F gene of Neurospora.

The function of the qa-1F positive regulatory gene of Neurospora has been studied by mapping the initiation sites for transcription of the clustered qa structural genes in wild type, in qa-1F mutants, and in cis-acting activator protein-independent mutants of qa-2 (qa-2ai mutants). Each structural gene under qa-1F control has two to four promoters. The qa-2ai mutations, which include point mutations and small (68-84 bp) duplications 5' to qa-2, allow qa-1F-independent transcription from surrounding qa promoters independently of the orientations and positions (up-stream or downstream) of teh mutations relative to the promoters. However, one subset of promoters was not reactivated by the enhancer-like elements created by these mutations, and qa-1F mutants selectively deficient in the activation of these promoters have been identified. Therefore, the qa-1F regulatory gene appears to control two types of promoters that have different requirements for activation.