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Ribulose 1,5-bisphosphate carboxylase was radiolabelled by in vitro translation, resulting in uniformly labelled ribulose 1,5-bisphosphate carboxylase, and also by reductive methylation. We investigated the degradation of the two forms of radiolabelled protein by natural bacterial populations. Although total hydrolysis of uniformly labelled protein and methylated protein was nearly equal, percent assimilation, respiration, and release as low-molecular-weight material were different. Radioactivity from uniformly labelled protein was approximately equally assimilated into cells, respired as H(2)O, and released as low-molecular-weight material, but radioactivity from the methylated protein was nearly all released as low-molecular-weight material, and little was assimilated or respired.

We have used a series of 30 DNA probes previously mapped to the long arm of the human Y chromosome, to screen a panel of 21 patients with structural abnormalities in Yq, by genomic blot hybridisation. The results have allowed us to construct a detailed map of interval 6 of the Y chromosome, in which 28 of the probes could be assigned to 14 sub-intervals within interval 6. Some probes detect two or more loci within this region, each of which has been localised. The same set of probes has been used to screen a panel of 19 chromosomally normal azoospermic men, two of whom have been found to carry microdeletions within this region. With the completion of this map we have been able accurately to localise these microdeletions within interval 6 and show that they do not overlap. We believe these microdeletions may disrupt the azoospermia factor (AZF) involved in spermatogenesis, and which is known to lie in this region. These results are an important step towards the localisation of the AZF locus.

The exteroceptive suppression (ES) of electrical activity in the temporal muscle is an inhibitory antinociceptive brain-stem reflex. We investigated whether aspirin can significantly modulate latencies or durations of the early (ES1) and late (ES2) exteroceptive suppression periods of electrical activity in the temporal muscle. Participating in the randomized double-blind crossover study were 20 patients with migraine without aura, 20 patients with tension-type headache, and 20 healthy subjects. ES1 and ES2 elicited by an electrical stimulus of 20 mA lasting 0.2 msec were recorded during maximal voluntary contraction of the mastication muscles before and 30 min after medication. In a randomized and double-blind fashion half of the subjects were given 1200 mg of aspirin in the form of an effervescent solution and the other half were given an identically tasting solution without aspirin. One week later the experiment was repeated with the substances exchanged in crossover fashion. The administration of placebo as well as aspirin caused a highly significant increase in ES1 duration (P less than or equal to 0.001). While aspirin caused a highly significant increase in ES2 duration (P less than or equal to 0.001) the taking of placebo showed no significant effect on ES2 duration. In giving aspirin as opposed to the placebo, there was a significant interaction between groups and drug effect on the latency of ES1; whereas in migraine patients and in patients with tension-type headache the latency of ES1 was reduced by administration of aspirin, it was increased in healthy subjects (P less than or equal to 0.05). Neither aspirin nor placebo significantly varied the ES2 latency.(ABSTRACT TRUNCATED AT 250 WORDS)

An experimental approach towards the molecular analysis of the male fertility function, located in interval 6 of the human Y chromosome, is presented. This approach is not based on the knowledge of any gene product but on the assumption that the functional DNA structure of male fertility genes, evolutionary conserved with their position on the Y chromosome, may contain an evolutionary conserved frame structure or at least conserved sequence elements. We tested this hypothesis by using dhMiF1, a fertility gene sequence of the Y chromosome of Drosophila hydei, as a screening probe on a pool of cloned human Y-DNA sequences. We were able to select 10 human Y-DNA sequences of which 7 could be mapped to Y interval 6 (the pY6H sequence family). Since the only fertility gene of the human Y chromosome is mapped to the same Y interval, our working hypothesis seems to be strongly supported. Most interesting in this respect is the isolation of the Y-specific repetitive pY6H65 sequence. The pY6H65 locus extends to a length of at least 300 kb in Y interval 6 and has a locus-specific repetitive sequence organization, reminiscent of the functional DNA structure of Y chromosomal fertility genes of Drosophila. We identified the simple sequence family (CA)n as one sequence element conserved between the Drosophila dhMiFi fertility gene sequence and the homologous human Y-DNA sequences.

HOT1 is a cis-acting recombination-stimulatory sequence isolated from the rDNA repeat unit of yeast. The ability of HOT1 to stimulate mitotic exchange appears to depend on its ability to promote high levels of RNA polymerase I transcription. A qualitative colony color sectoring assay was developed to screen for trans-acting mutations that alter the activity of HOT1. Both hypo-recombination and hyper-recombination mutants were isolated. Genetic analysis of seven HOT1 recombination mutants (hrm) that decrease HOT1 activity shows that they behave as recessive nuclear mutations and belong to five linkage groups. Three of these mutations, hrm1, hrm2, and hrm3, also decrease rDNA exchange but do not alter recombination in the absence of HOT1. Another mutation, hrm4, decreases HOT1-stimulated recombination but does not affect rDNA recombination or exchange in the absence of HOT1. Two new alleles of RAD52 were also isolated using this screen. With regard to HOT1 activity, rad52 is epistatic to all four hrm mutations indicating that the products of the HRM genes and of RAD52 mediate steps in the same recombination pathway. Finding mutations that decrease both the activity of HOT1 and exchange in the rDNA supports the hypothesis that HOT1 plays a role in rDNA recombination.

We examined the genetic control of the activity of HOT1, a cis-acting recombination-stimulatory sequence of Saccharomyces cerevisiae. Mutations in RAD1 and RAD52 decrease the ability of HOT1 to stimulate intrachromosomal recombination while mutations in RAD4 and RAD50 do not affect HOT1 activity. In rad1 delta strains, the stimulation of excisive recombination by HOT1 is decreased while the rate of gene replacement is not affected. In rad52-8 strains the ability of HOT1 to stimulate both excisive recombination and gene replacement is decreased. All of the recombinants in the rad52-8 strains that would be categorized as gene replacements based on their phenotype are diploids apparently derived by endomitosis and excisive recombination. Studies on rad1 delta rad52-8 strains show that these mutations interact synergistically in the presence or absence of HOT1, resulting in low levels of recombination. The rate of gene replacement but not excisive recombination is stimulated by HOT1 in rad1 delta rad52-8 strains. Taken together, the results show that HOT1 stimulates exchange using multiple recombination pathways. Some of the activity of HOT1 is RAD1-dependent, some is RAD52-dependent, and some requires either RAD1 or RAD52 as suggested by the synergistic interaction found in double mutant strains. There is also a component of HOT1 activity that is independent of both RAD1 and RAD52.

The effects of guanine nucleotides on binding of 8-cyclopentyl-1,3-[3H]dipropylxanthine ([3H]DPCPX), a highly selective A1 adenosine receptor antagonist, have been investigated in rat brain membranes and solubilized A1 receptors. GTP, which induces uncoupling of receptors from guanine nucleotide binding proteins, increased binding of [3H]DPCPX in a concentration-dependent manner. The rank order of potency for different guanine nucleotides for increasing [3H]DPCPX binding was the same as for guanine nucleotide-induced inhibition of agonist binding. Therefore, a role for a guanine nucleotide binding protein, e.g., Gi, in the regulation of antagonist binding is suggested. This was confirmed by inactivation of Gi by N-ethylmaleimide (NEM) treatment of membranes, which resulted in an increase in [3H]DPCPX binding similar to that seen with addition of GTP. Kinetic and equilibrium binding studies showed that the GTP- or NEM-induced increase in antagonist binding was not caused by an affinity change of A1 receptors for [3H]DPCPX but by an increased Bmax value. Guanine nucleotides had similar effects on membrane-bound and solubilized receptors, with the effects in the solubilized system being more pronounced. In the absence of GTP, when most receptors are in a high-affinity state for agonists, only a few receptors are labeled by [3H]DPCPX. It is suggested that [3H]DPCPX binding is inhibited when receptors are coupled to Gi. Therefore, uncoupling of A1 receptors from Gi by guanine nucleotides or by inactivation of Gi with NEM results in an increased antagonist binding.

Many genetic studies have shown that the frequency of homologous recombination depends largely on the distance in which recombination can occur. We have studied the effect of varying the length of duplicated sequences on the frequency of mitotic intrachromosomal recombination in Saccharomyces cerevisiae. We find that the frequency of recombination resulting in the loss of one of the repeats and the intervening sequences reaches a plateau when the repeats are short. In addition, the frequency of recombination to correct a point mutation contained in one of these repeats is not proportional to the size of the duplication but rather depends dramatically on the location of the mutation within the repeated sequences. However, the frequency of mitotic interchromosomal reciprocal recombination is dependent on the distance separating the markers. The difference in the response of intrachromosomal and interchromosomal mitotic recombination to increasing lengths of homology may indicate there are different rate-limiting steps for recombination in these two cases. These findings have important implications for the maintenance and evolution of duplicated sequences.

Special mechanisms for stimulating recombination among the nearly identical repeat units of certain multigene families may exist in order to maintain their sequence homogeneity. We have found evidence for such a recombination-stimulating activity in the tandemly repeated ribosomal RNA genes of yeast. A fragment of the yeast ribosomal DNA (rDNA), containing the 5S gene, nontranscribed spacer DNA, and part of the 25S gene, causes a localized stimulation of recombination when inserted at novel locations in the yeast genome. The rDNA fragment stimulates both interchromosomal and intrachromosomal mitotic recombination but not meiotic recombination. To stimulate mitotic recombination, the fragment must act on both copies of the recombining gene. Furthermore, the rDNA fragment stimulates exchange only when inserted with the 5S gene proximal to, and the 25S gene distal to, the recombining alleles.

A DNA sequence (HOT1) from the repeated ribosomal RNA gene cluster of Saccharomyces cerevisiae can stimulate genetic exchange when inserted at novel locations in the yeast genome. Localization of the sequences required for HOT1 activity demonstrates that two noncontiguous fragments of DNA are required for the stimulation of recombination. One of these fragments contains the transcription initiation site for the major 35S ribosomal RNA precursor. The other contains an enhancer of RNA polymerase I transcription. We suggest that transcription by RNA polymerase I initiating in the inserted rDNA and proceeding through the adjacent sequences is responsible for the stimulation of exchange. Consistent with this interpretation, insertion of the putative termination site for RNA polymerase I transcription between HOT1 and the adjacent recombining DNA abolishes the recombination stimulation. Transcription through both copies of the homologous recombining sequences appears to be necessary for enhanced exchange.