Characterization of the aspartate carbamoyltransferase fragment generated by protease action on the pyrimidine-3 gene product of Neurospora crassa.

The molecular weight of the fragment of aspartate carbamoyltransferase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) of Neurospora crassa following proteolysis was found to be 1.0-10(5) (aspartate carbamoyltransferase-L). It differs from the native form of the enzyme (aspartate carbamoyltransferase-N, 6.5-10(5)) in several respects. It has a lower V, has a much greater affinity (approx. 3-fold) for L-aspartate, and is strongly activated by glycine. Both forms of aspartate carbamyoltransferase have a pH optimum of approx. 9.5, and they exhibit similar affinities for carbamoyl phosphate.

Association study of dopamine receptor gene polymorphisms with drug-induced hallucinations in patients with idiopathic Parkinson's disease.

Some patients with idiopathic Parkinson's disease experience hallucinations as a result of treatment with levodopa and dopamine agonists. There is evidence for some heterogeneity in these hallucinating patients based on duration of Parkinson's disease at onset of hallucinations. We compared the frequency of polymorphisms in the dopamine D2 and D3 receptor genes between patients with drug-induced hallucinations and non-hallucinating patients. Two polymorphisms close to DRD2 and one in DRD3 were studied. No association was found with the whole group of hallucinating patients and their controls. However, an association was found with late-onset hallucinations and the C allele of the TaqIA polymorphism, 10.5 kb 3' to DRD2. This polymorphism may be in linkage disequilibrium with a mutation in DRD2 or a nearby gene that predisposes to drug-induced hallucinations which occur later in the course of idiopathic Parkinson's disease.

Association of mu-opioid receptor subunit gene and idiopathic generalized epilepsy.

OBJECTIVE: To replicate and extend the previously reported association between the opioid receptor mu subunit gene (OPRM1) and idiopathic absence epilepsy (IAE), using a sample of 230 probands with idiopathic generalized epilepsy (IGE). BACKGROUND: In humans and in animal models, several lines of evidence implicate opioid receptors with seizures. The G118 allele of OPRM1 was associated with IAE (p = 0.019). METHODS: Three single nucleotide polymorphisms (SNP) of OPRM1 were investigated by association studies with IGE using a case/control design, one of which also used a within-family design. RESULTS: Association was found for G118 with IGE (p = 0.00027, odds ratio [OR] = 1.86), replicating the previous association. Within-family tests of linkage and association (haplotype-based haplotype relative risk and transmission disequilibrium test) confirmed this result. Further evidence for involvement of OPRM1 in IGE was provided by an association with G-172T, located in the 5' untranslated region (p = 0.0015, OR = 2.36). Haplotypes of the two SNPs were associated with IGE with a greater level of significance (p = 0.000087) suggesting that both SNPs might be in linkage disequilibrium with a single functional variant. Analysis of the results by subgroups of IGE showed association with all subgroups tested. CONCLUSIONS: These results confirm the previous association and support the hypothesis of a role for OPRM1 in IGE, including absence syndromes. However, the authors found no evidence for a specific association between OPRM1 and idiopathic absence epilepsy. The data suggest that the functional variant predisposing to IGE is located within 60kb of exon 1.

Failure to replicate association between the gene for the neuronal nicotinic acetylcholine receptor alpha 4 subunit (CHRNA4) and IGE.

The gene for the neuronal nicotinic acetylcholine receptor alpha4 subunit (CHRNA4) was identified as a gene underlying a rare idiopathic partial epilepsy syndrome in humans, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). In a recent study, one of four silent polymorphisms (594 C/T) in CHRNA4 showed association with the common subtypes of idiopathic generalised epilepsy (IGE). In the present study, three of these polymorphisms were investigated for association in 182 Caucasian patients with IGE, but not categorised by subtype. They were compared with 178 controls in a case/control study. Further analyses were performed using a family-based design. None of the three polymorphisms exhibited any association with IGE. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:814-816, 2000.

Polymorphisms in the genes for mGluR types 7 and 8: association studies with schizophrenia.

A dysfunctional glutamatergic system has been implicated in the pathophysiology of schizophrenia. The group III metabotropic glutamate receptor (mGluR) types 7 and 8 presynaptically inhibit glutamate release, thereby modulating glutamatergic transmission in the brain. We conducted association studies to investigate the novel Tyr433Phe (mGluR7) variant and the 2846-C/T (mGluR8) polymorphism in schizophrenia. Both variants, present at high frequencies, failed to demonstrate any significant association with schizophrenia (mGluR7 [Tyr433Phe] allele: P=0.33; genotype: P=0.63; mGluR8 [2846-C/T] allele: P=0.72; genotype: P=0.63).

Expression of a novel splice variant of human mGluR1 in the cerebellum.

We have isolated clones of a novel splice variant of metabotropic glutamate receptor type 1 (mGluR1) from a human cerebellum cDNA library. Translation of this variant, mGluR1g would result in the addition of just one amino acid after the exon/intron boundary where the other splice variants diverge. RNA dot blot analysis using an mGluR1g-specific probe demonstrated expression in the cerebellum and also high levels in the kidney. Northern blotting using the same probe showed expression of a 4 kb transcript in the cerebellum. In situ hybridization studies in the cerebellum showed that mGluR1g mRNA is only expressed in granule cells, compared with mGluR1a/b mRNA which is also found in Purkinje cells and basket cells. Transcripts of the analogous splice variant are also present in rat brain mRNA.

No association found between polymorphisms in genes encoding mGluR7 and mGluR8 and idiopathic generalised epilepsy in a case control study.

The genes of two group III metabotropic glutamate receptors, mGluR7 and 8, are candidate susceptibility genes for epilepsy. The Tyr433Phe polymorphism of mGluR7 and a novel polymorphism in the mGluR8 gene located 29 bp after the termination codon (2756C/T) were investigated in case control association studies performed on DNA from more than 100 patients with idiopathic generalised epilepsy (IGE). No significant association was found with IGE for either polymorphism.

Suggestive evidence for association of two potassium channel genes with different idiopathic generalised epilepsy syndromes.

Several potassium channel genes have been implicated in epilepsy. We have investigated three such genes, KCNJ3, KCNJ6 and KCNQ2, by association studies using a broad sample of idiopathic generalised epilepsy (IGE) unselected by syndrome. One of the two single nucleotide polymorphisms (SNPs) examined in one of the inward rectifying potassium channel genes, KCNJ3, was associated with IGE by genotype (P=0.0097), while its association by allele was of borderline significance (P=0.051). Analysis of the different clinical subgroups within the IGE sample showed more significant association with the presence of absence seizures (P=0.0041) and which is still significant after correction for multiple testing. Neither SNP in the other rectifying potassium channel gene, KCNJ6, was associated with IGE or any subgroup. None of the three SNPs in the voltage-gated potassium channel gene, KCNQ2, was associated with IGE. However, one SNP was associated with epilepsy with generalised tonic clonic seizures only (P=0.016), as was an SNP approximately 56 kb distant in the closely linked nicotinic acetylcholine gene CHRNA4 (P=0.014). These two SNPs were not in linkage disequilibrium with each other, suggesting that if they are not true associations they have independently occurred by chance. Neither association remains significant after correcting for multiple testing.

Polymorphism analysis of JRK/JH8, the human homologue of mouse jerky, and description of a rare mutation in a case of CAE evolving to JME.

Disruption of the function of the mouse jerky gene by transgene insertion causes generalized recurrent seizures reminiscent of human idiopathic generalized epilepsy (IGE). A human homologue, JRK/JH8, has been cloned, which maps to 8q24, a chromosomal region associated with several forms of IGE. JRK/JH8 is, therefore, a candidate locus for at least some forms of IGE. We report corrected cDNA sequences and extended open reading frames for the mouse jerky and human JRK/JH8 genes, which add 48 amino acids to the N-terminus of the Jerky protein and which extends the region of homology with the N-terminal DNA-binding domain of the centromere-binding protein, CENP-B. Systematic sequencing of the coding region of the extended JRK/JH8 gene identified single nucleotide polymorphisms that define three haplotypes, which were used for association studies in patients with idiopathic generalized epilepsy. We report one subject with childhood absence epilepsy (CAE) that evolved to juvenile myoclonic epilepsy (JME) that has a unique de novo mutation that results in a non-conservative amino acid change at a potential protein glycosylation site. Familial analysis supports a causal role for this mutation in the disease.

Protective surface antigen P69 of Bordetella pertussis: its characterization and very high level expression in Escherichia coli.

The surface antigen, P69 of Bordetella pertussis, an N-terminal fragment of the precursor protein, P93, is likely to be an important component of future subunit vaccines against whooping cough. We have expressed several defined N-terminal fragments of P93 in E. coli and compared their electrophoretic mobilities with that of purified P69 from B. pertussis. These experiments show that P69 is considerably smaller than the 69 kD originally estimated from its gel mobility and is probably 60.4 kD in size. Our initial plasmids expressed only very low levels of this antigen. We diagnosed the limiting factor to be a poor ribosome binding site (RBS) by demonstrating a large stimulation of expression on a two-cistron plasmid. The limitation of expression could be completely overcome by only two base changes close to the initiation codon, resulting in a further increase in expression of P69 at levels to 30-40% total cell protein. Although the protein accumulated as insoluble inclusion bodies, it could be solubilized by guanidinium chloride.

Use of the nirB promoter to direct the stable expression of heterologous antigens in Salmonella oral vaccine strains: development of a single-dose oral tetanus vaccine.

Plasmid pTETnir15, which directs the expression of the non-toxic immunogenic fragment C of tetanus toxin from the anaerobically inducible nirB promoter, was introduced into the Salmonella typhimurium aroA aroD live oral vaccine strain BRD509. The resulting strain, designated BRD847, was used to vaccinate orally BALB/c mice and was tested for plasmid stability and its ability to protect against a lethal tetanus toxin challenge. pTETnir15 was stably inherited by bacteria growing or persisting in the tissues of immunized mice whereas another BRD509 derivative, designated BRD753, harboring plasmid pTET85 which directs fragment C expression from the tac promoter, was highly unstable. Mice immunized with a single oral dose of BRD847 developed high levels of circulating anti-fragment C antibodies and were solidly protected against tetanus toxin challenge. Mice immunized with a single oral dose of BRD753 developed no detectable anti-fragment C antibodies. After boosting, antibodies were detected, but the mice were only partially protected against tetanus toxin challenge. Thus the use of an in vivo inducible promoter such as nirB may be a generally applicable approach to obtaining the stable in vivo expression of heterologous antigens in Salmonella vaccine strains.

High level heterologous expression in E. coli using mutant forms of the lac promoter.

Single base deletions in the lac promoter which reduced the 18bp spacing between the -35 and -10 homology regions to 17bp, increased the strength of the promoter. A single base substitution (T----G) in the -35 region to generate the consensus sequence TTG-ACA increased the strength further and no longer required a 17bp spacing. The mutated lac promoter was as powerful as a shorter form of the tac promoter which lacked two AT-rich regions upstream of the -35 region, and expressed the P69 surface antigen (pertactin) of Bordetella pertussis to 30-40% total cell protein and tetanus toxin fragment C to 16-20% total cell protein.

Identification of a class of lysines within the non-specific DNA-binding site of RNA polymerase core enzyme from Escherichia coli.

The imido ester, methyl acetimidate, which specifically amidinates lysine residues, modifies RNA polymerase core enzyme, leading to rapid loss of activity. Calf thymus DNA partially protects the enzyme against this inactivation, an effect which disappears at high salt concentration. DNA protects 17 +/- 6 lysines from amidination at low salt concentration. The dependence of amidination on methyl acetimidate concentration is examined in the presence of DNA at high and low salt concentration. Analysis of the data suggests a class of approximately 12 lysines which are protected by DNA, consistent with the above estimate. These lysines are approximately 5--10-fold more reactive than most other available lysine residues.

The location of the feedback-specific region with the pyrimidine-3 locus of Neurospora crassa.

The purimidine-3 locus of Neurospora crassa specifies two enzyme activities, pyrimidine-specific carbamyl phosphate synthetase (CPSpyr) and aspartate transcarbamylase (ATC). ATC is translationally distal. CPSpyr, but not ATC, is subject to feedback inhibition by uridine triphosphate (UTP). To investigate the location of the feedback-specific region within the locus, inhibition of a number of pyr-3 alleles by UTP was investigated. All CPS+ ATC- polar alleles, revertants of CPS- ATC- polar alleles, and 5-fluorouracil-resistant mutants had normal UTP response. The location of the feedback-specific region is in or close to the CPS-specific region.

Glutamine utilization in both the arginine-specific and pyrimidine-specific carbamoyl phosphate synthase enzymes of eurospora crassa.

As glutamine-dependent carbamoyl phasphate synthetase (CPS) activity in some organisms is composed of a glutaminase and an ammonium-dependent CPS, CPS- mutants in Neurospora crassa were examined for glutamine- and ammonium-dependent CPS activities. No evidence was found that the genetic location of these two functions were separable. This is discussed with reference to the close genetic proximity of the CPSpyr and aspartate carbamoyltransferase (ACT) structural gene (pyr-3) and the arg-2 gene which appears to specify a subunit responsible for glutamine utilisation in CPSarg.

Properties of methyl acetimidate and its use as a protein-modifying reagent.

The rate of hydrolysis of the imido ester methyl acetimidate and its rate of amidination of denatured aldolase were investigated under different conditions of temperature, pH and ionic strength. Both rate constants increase greatly with temperature, whereas ionic strength has no effect on either. The effect of pH is more complex. Between pH 6.8 and 8.8 the rate of hydrolysis decreases and the rate of amidination increases. These results are discussed in terms of the reaction mechanisms involved.

The use of two-cistron constructions in improving the expression of a heterologous gene in E. coli.

Many heterologous genes when cloned into bacterial expression vectors are poorly expressed because of an inefficient ribosome binding site (RBS). We have constructed a plasmid which expresses human gamma-interferon (gamma-IF), where the level of expression is limited by the RBS. Expression was increased by placing the gamma-IF sequence immediately downstream of a small translated sequence. The production of gamma-IF was dependent upon the efficiency of translation of this upstream cistron and could be increased to very high levels. The same upstream cistron would greatly improve the expression of gamma-IF in a plasmid where the RBS was very poor due to inhibitory secondary structure at the 5' end of its mRNA. However, it would not improve the efficiency of a poor RBS containing a weak Shine-Dalgarno sequence. The general utility of the two-cistron expression strategy to diagnose a weak RBS is discussed.

A possible model for the structure of the Neurospora carbamoyl phosphate synthase-aspartate carbamoyl transferase complex enzyme.

The pyrimidine-3 locus of Neurospora crassa specifies a multienzyme complex comprising pyrimidine-specific carbamoyl phosphate synthase (CPSpyr) and aspartate carbamoyl transferase (ACT). It appears to be divided into a translationally proximal CPS-specific region and a distal ACT-specific region. Levels of complementation for ACT activity between pairs of four pyr-3 CPS+ ACT- mutants showed a range from 12% to 68% of the wild-type level of the enzyme. This is interpreted as interallelic complementation, contradicting certain earlier suggestion of two dissimilar ACT subunits. Proteolysis of an extract from a heterokaryon formed from two of the above CPS+ ACT- alleles (alpha and beta) did not lead to loss of ACT activity, but led to the formation of a fragment with ACT activity with a similar molecular weight (92,000 daltons) to that produced in extracts of wild type strain. The pyr-3 polar mutant 43-174 which is enzymatically CPS+ ACT- and which fails to complement with any other CPS+ ACT- alleles, thus suggesting its location towards the proximal end of the ACT region, has CPS activity associated with a form of 180,000 daltons molecular weight. These findings are used to contruct a model for structure of the native enzyme complex.