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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)

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The qa-iF regulatory gene of Neurospora crassa encodes an activator protein required for quinic acid induction of transcription in the qa gene cluster. This activator protein was expressed in insect cell culture with a baculovirus expression vector. The activator binds to 13 sites in the gene cluster that are characterized by a conserved 16-base-pair sequence of partial dyad symmetry. One site is located between the divergently transcribed qa-iF and qa-IS regulatory genes, corroborating prior evidence that qa-iF is autoregulated and controls expression of the qa-iS repressor. Multiple upstream sites located at variable positions 5' to the qa structural genes appear to allow for greater transcriptional control by qa-IF. Full-length and truncated activator peptides were synthesized in vitro, and the DNA-binding domain was localized to the first 183 amino acids. A 28-amino acid sequence within this region shows striking homology to N-terminal sequences from other lower-eucaryotic activator proteins. A q4-1F(Ts) mutation is located within this putative DNA-binding domain.

Several reports have been published on the use of polymorphisms found in the human hemoglobin genes as a means for prenatal diagnosis of sickle cell anemia. The disadvantages ofthis approach reside in its limited application and the need for family analysis. Here we report that, by use of restriction endonuclease Dde I and diazobenzyloxymethyl-paper transfer procedures, a direct analysis can be made. Individuals with normal hemoglobin (AA) show two bands (175 and 201 base pairs) complementary to a 5'-specific 13-globin gene probe. Sickle cell trait individuals (AS) exhibit an additional band (376 base pairs). Individuals with sickle cell anemia (SS) show the band at 376 base pairs with a concomitant loss of the 175-base pair band. We interpret these changes in banding pattern to be the result of the elimination of a restriction site for Dde I in the altered codon associated with the sickle cell allele. Because an analysis can be performed on as little as 20 jig of cellular DNA, the application to prenatal diagnosis of sickle cell anemia should be possible.Recombinant DNA techniques coupled with blot hybridization analysis have proven to be valuable tools for studying the molecular basis of hemoglobinopathies. Various researchers have used blot hybridization to confirm that 8,f3thalassemia (1-5) and hereditary persistence of fetal hemoglobin (1)(2)(3)(4)(5)(6) are the result of gene deletions, whereas a-thalassemia (7-9) and 13-thalassemia (10-12) are due to both gene deletions and point mutations. One study has shown that at least one case of 8-thalassemia is probably due to a base mutation (13). These studies have also been extended to the clinical setting as methods for prenatal diagnosis of various genetic hematological conditions (14-19). Kan and Dozy have reported (20) the finding of a polymorphism for a Hpa I restriction endonuclease site in American Blacks 3' to the ,-globin gene, which was shown to have a 60% association with the sickle cell allele. From their studies, they estimated that blot hybridization using this polymorphism alone could be successfully used for prenatal diagnosis of a sickle cell anemia in 36% of couples at risk. Phillips et al. (21) have combined the Hpa I analysis with a second polymorphism found in the y-globin genes (22). In so doing, they have reported (21) an extension of blot hybridization for prenatal diagnosis of sickle cell anemia to over 80% of the couples at risk. However, both require family studies in order to establish the association ofthe polymorphic sites with the sickle cell allele. This limited application is a major disadvantage of these procedures.A direct analysis of the sickle cell anemia should be possible by use of a restriction enzyme whose recognition sequence is created or eliminated by the sickle cell mutation. This approach would not require family studies and should be useful for all couples at risk.Nienhuis has proposed such a direct analysis with restriction endonuclease Mnl I (23). However, efforts in various laboratories have failed t...