The nucleotide sequence of Saccharomyces cerevisiae chromosome XVI.

The nucleotide sequence of the 948,061 base pairs of chromosome XVI has been determined, completing the sequence of the yeast genome. Chromosome XVI was the last yeast chromosome identified, and some of the genes mapped early to it, such as GAL4, PEP4 and RAD1 (ref. 2) have played important roles in the development of yeast biology. The architecture of this final chromosome seems to be typical of the large yeast chromosomes, and shows large duplications with other yeast chromosomes. Chromosome XVI contains 487 potential protein-encoding genes, 17 tRNA genes and two small nuclear RNA genes; 27% of the genes have significant similarities to human gene products, and 48% are new and of unknown biological function. Systematic efforts to explore gene function have begun.

The modulation of brain dopamine and GABAA receptors by estradiol: a clue for CNS changes occurring at menopause.

1. Tardive dyskinesia is more important in postmenopausal women than men of comparable age and a peak of first episodes of schizophrenia is observed in postmenopausal women. The effect of ovariectomy (2 weeks or 3 months) in rats was investigated as a model of decreased gonadal function associated with menopause. 2. Frontal cortex D1 receptor density and affinity were similar in intact male compared to intact female rats and progressively decreased in density with time after ovariectomy, with no change of affinity. Striatal D1 and D2 receptors also decreased in density after ovariectomy for both receptor subtypes, with no change of affinity. Striatal D1 receptor density and affinity were similar in intact male and female rats, whereas the density of D2 receptors was higher in females. Treatment with estradiol for 2 weeks restored the D2 but not the D1 receptor changes. 3. In the substantia nigra pars reticulata, striatum, nucleus accumbens, and entopeduncular nucleus, a progressive increase in [3H]flunitrazepam specific binding associated with GABAA receptors was observed as a function of time following ovariectomy; this was corrected with estradiol treatment. In contrast, the opposite was observed for [3H] flunitrazepam binding in the globus pallidus, where ovariectomy decreased binding, which was corrected with estradiol replacement therapy. 4. Low prefrontal cortex dopamine activity with implications of D1 receptors in negative symptoms of schizophrenia is hypothesized. Furthermore, GABAergic overactivity in the internal globus pallidus-substantia nigra pars reticulata complex is hypothesized in tardive dyskinesia. 5. The present data suggest that gonadal hormone withdrawal by reducing brain dopamine receptors and producing an imbalance of GABAA receptors in the output pathways of the striatum may predispose to schizophrenia and dyskinesia.

Prp20, the Saccharomyces cerevisiae homolog of the regulator of chromosome condensation, RCC1, interacts with double-stranded DNA through a multi-component complex containing GTP-binding proteins.

Prp20, a homolog to the mammalian negative regulator of chromosome condensation, RCC1, is retained on double-stranded (ds) DNA-cellulose when extracts are prepared from asynchronously growing wild-type yeast strains. Conversely, neither Prp20 from ts mutant cell extracts nor wt yeast Prp20 produced in Escherichia coli, bind to dsDNA-cellulose. In vitro reconstitution assays using E. coli-expressed Prp20 and inactivated ts mutant extracts of prp20-1 reveal that the Prp20 protein requires the assistance of other proteins in the cell extract to promote its binding to dsDNA. Immunoprecipitations and sizing-column-chromatography indicate that the Prp20 protein binds to the dsDNA column through a multicomponent complex composed of six to seven proteins, which has a collective molecular mass greater than 150,000 Da. At least three of the members of this Prp20 complex will bind GTP in vitro. Moreover, the Prp20 complex is shown to specifically lose its ability to bind dsDNA during the DNA replication phase of the cell cycle. This loss of dsDNA binding during the S phase of the cell cycle does not affect the proper organization of the nucleoplasm and appears to be reversed before the cell enters mitosis.

GSP1 and GSP2, genetic suppressors of the prp20-1 mutant in Saccharomyces cerevisiae: GTP-binding proteins involved in the maintenance of nuclear organization.

The temperature-sensitive mutation prp20-1 of Saccharomyces cerevisiae exhibits a pleiotropic phenotype associated with a general failure to maintain a proper organization of the nucleus. Its mammalian homolog, RCC1, is not only reported to be involved in the negative control of chromosome condensation but is also believed to assist in the coupling of DNA replication to the entry into mitosis. Recent studies on Xenopus RCC1 have strongly suggested a further role for this protein in the formation or maintenance of the DNA replication machinery. To elucidate the nature of the various components required for this PRP20 control pathway in S. cerevisiae, we undertook a search for multicopy suppressors of a prp20 thermosensitive mutant. Two genes, GSP1 and GSP2, were identified that encode almost identical polypeptides of 219 and 220 amino acids. Sequence analyses of these proteins show them to contain the ras consensus domains involved in GTP binding and metabolism. The levels of the GSP1 transcript are about 10-fold those of GSP2. As for S. cerevisiae RAS2, GSP2 expression exhibits carbon source dependency, while GSP1 expression does not. GSP1 is an essential gene, and GSP2 is not required for cell viability. We show that GSP1p is nuclear, that it can bind GTP in an in vitro assay, and finally, that a mutation in GSP1p which activates small ras-like proteins by increasing the stability of the GTP-bound form causes a dominant lethal phenotype. We believe that these two gene products may serve in regulating the activities of the multicomponent PRP20 complex.

Correlation of functional recovery after a 6-hydroxydopamine lesion with survival of grafted fetal neurons and release of dopamine in the striatum of the rat.

Female rats were lesioned with 6-hydroxydopamine in the left substantia nigra. At least two weeks later they were tested with amphetamine (5 mg/kg, s.c.) and apomorphine (0.25 mg/kg, s.c.). A cell suspension from the ventral mesencephalon of rat embryos was distributed in three sites in a triangular fashion in the center of the denervated striatum. The amphetamine test was then repeated every month for six months. The pattern of circling to amphetamine before the graft was strictly ipsiversive in all animals. From the first month we observed a progressive change and three patterns of rotation could be observed. In 21% of animals, the total number of ipsiversive turns in 90 min actually increased but during the first 20 min the animals turned contralaterally to the lesion (and to the graft). In 38% of animals, the total number of turns switched from ipsiversive to contraversive with the animals turning initially toward the intact side and during the second half of the test toward the lesion. Finally 41% of rats progressively switched to turning only toward the intact side. In all cases, maximal contraversive turning occurred during the initial 20 min. In these rats, tyrosine hydroxylase-positive cells were detected mainly in the dorsal striatum with a few in the central portion. Moreover there was a strong correlation between the number of surviving grafted neurons and the growth of their fiber into the host striatum and the extent of recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

[Animal models of tardive dyskinesia]. / Les modèles animaux de la dyskinésie tardive.

It is possible to induce in monkeys abnormal movements of the mouth and tongue resembling strikingly tardive dyskinesia. Such movements can be induced by chronic treatment with neuroleptics or by lesions placed in the habenular interpeduncular tract or nucleus parafascicularis thalami. Dopaminergic D1 and D2 receptors are involved in the modulation of such movements.

Behavioral and biochemical evidence for a different effect of repeated administration of L-dopa and bromocriptine on denervated versus non-denervated striatal dopamine receptors.

The effect of repeated administration of bromocriptine and L-3,4-dihydroxyphenylalanine (L-DOPA) was studied behaviorally and biochemically in rats with a unilateral lesion of the nigrostriatal pathway. Groups of rats injected eight times with bromocriptine or L-DOPA significantly increased their contraversive circling. Rats receiving only two injections of bromocriptine did not. Animals receiving two injections of L-DOPA showed a slight but significant increase in circling. The affinity of the binding of [3H]spiperone to the dopamine receptors was unchanged by the lesion or the treatments, while the density of the binding was significantly modified. Chronic treatment with bromocriptine induced a significant decrease in the density of D2 dopamine receptors in the intact striata, while on the lesioned side, it remained unchanged. By contrast, chronic administration of L-DOPA induced a significant increase in density of the striatal dopamine receptors in the lesioned striata in addition to that caused by denervation, while the decrease on the intact side was not significant. It seems that contrary to the intact striatum, the lesioned side had a defective down-regulation mechanism in response to chronic treatment with a dopamine agonist. The results also show that L-DOPA was more potent than bromocriptine in inducing agonist supersensitivity in a denervated striatum. This may explain why chronic treatment with bromocriptine has a lesser tendency to induce dyskinesia in patients with Parkinson's disease.

Impaired regulation of neurohypophyseal vasopressin secretion in rats treated neonatally with monosodium-L-glutamate.

Monosodium-L-glutamate (MSG) lesioning of the arcuate nucleus (ARC) was utilized to elucidate the importance of its dopaminergic system for regulation of neurohypophyseal vasopressin (AVP) secretion. Water deprivation in control animals elicited a rise in plasma AVP and depletion of neurohypophyseal AVP, at unchanged AVP contents of the paraventricular (PVN) and supraoptic (SON) nuclei. Dopaminergic activity was markedly stimulated in the PVN and ARC. Neonatal MSG treatment resulted in elevated plasma AVP levels at reduced neurohypophyseal AVP due to diminished dopaminergic input from the ARC. When MSG treated animals were subjected to dehydration, none of the changes in plasma/neurohypophyseal AVP and dopamine metabolism in the PVN and ARC observed in control animals were seen. These results suggest that the ARC plays an important role in the dopaminergic control of neurohypophyseal AVP secretion.

Action of 5-hydroxytryptamine, substance P, thyrotropin-releasing hormone and clonidine on motoneurone excitability.

We have investigated the influence on the excitability of lumbar motoneurons of 5-hydroxytryptamine (5-HT), substance P and thyrotropin releasing hormone (TRH), three substances which coexist in the same bulbospinal descending pathway and end in large part around motoneurons. We have also studied the effect of clonidine, an alpha 2 noradrenergic agonist. This was done in spinalized rats (T5) treated three weeks before with 5-7-dihydroxytryptamine. Under those circumstances 5-HTP (I.P.), 5-HT (intrathecally) TRH (I.P. or I.T.) and substance P (I.T.) all elicited a strong excitation of motoneurons as measured by integrated EMG of the hindlimb muscles. Substance P reduced by almost half the subsequent response to 5-HTP, 1 hour and 24 hours later. TRH given acutely did not modify the response to 5-HTP but given chronically for twenty one days by means of Alzet minipump, markedly increased the response to 5-HTP. Clonidine by itself decreased the excitability of motoneurons and antagonized the excitatory effect of 5-HTP and TRH. In a pilot trial, cyproheptadine, a 5-HT antagonist was shown to decrease the manifestations of spasticity in patients with a partial spinal lesion. Clonidine also appears to be of potential use in the treatment of spasticity.

Estradiol, TRH and striatal dopaminergic mechanisms.

1. Estrogens have been shown to decrease the effect of apomorphine in a variety of animal behavioral models reflecting the sensitivity of striatal and mesolimbic dopamine receptors. 2. These include circling, and locomotor activity, in rats and suppression of midbrain tremor as well as lingual dyskinesia in monkeys. 3. Estradiol also increases the haloperidol-induced catalepsy in rats. Moreover estradiol increases 3H spiroperidol specific binding in the rat striatum and potentiates the increase caused by haloperidol or denervation with 6-hydroxydopamine. 4. These findings point to an action of estradiol similar to a week neuroleptic. 5. Thyrotropin-releasing hormone when injected into the head of the caudate nucleus in cats induces a head turning response which may be ipsilateral or contralateral depending upon the injection site. The response is similar to the effect of dopamine injected into the same site. 6. The effect of dopamine but not that of TRH is blocked by prior administration of haloperidol indicating that although TRH has a dopamine-like action in the caudate nucleus, it is not mediated via the dopamine terminals or the dopamine receptors.

Effect of neonatal thyroid deficiency on the catecholamine, substance P, and thyrotropin-releasing hormone contents of discrete rat brain nuclei.

The effects of neonatal thyroidectomy and thyroid hormone replacement therapy on the development of catecholamine-, TRH-, and substance P-containing neurons in discrete rat brain nuclei were studied. Newborn male rats were rendered hypothyroid by the injection of 125 muCi 131I and, after 45 days, were compared with normal littermate controls and 131I-injected animals subsequently maintained on T4 injections. The peptide or catecholamine content of discrete brain nuclei removed by punches of frozen brain slices was measured by RIA or radioenzymatic assay, respectively. The success of the thyroidectomy was verified by criteria of weight, length, plasma T4, and pituitary GH content. Animals receiving T4 replacement therapy were indistinguishable from normal littermates. Substance P was measured in 32 different brain nuclei and was significantly increased in 19 of these areas in hypothyroid animals. No changes in norepinephrine were detected, and the dopamine content of all but 3 brain nuclei was increased by thyroidectomy. The TRH concentration was drastically reduced in the median eminence of hypothyroid animals and also changed in 3 other extrahypothalamic areas. All of the changes seen in catecholamine, TRH, and substance P distribution in hypothyroid animals were completely reversed by T4 replacement therapy. These results demonstrate changes in brain peptide neurotransmitters during the hypothyroid state and open new vistas for comprehension of biochemical mechanisms underlying central nervous system malfunction.