The gene encoding the 49-kDa subunit of RNA polymerase A in Saccharomyces cerevisiae has been identified by formation of a hybrid enzyme between the S. cerevisiae A49 subunit and Saccharomyces douglaui subunits based on a polymorphism existing between the subunits ofRNA polymerase A in these two species. The sequence of the gene reveals a basic protein with an unusually high lysine content, which may account for the affinity for DNA shown by the subunit. No appreciable homology with any polymerase subunits, enzymes, or transcription factors is found. Complete deletion of the single-copy RPA49 gene leads to viable but slowly growing colonies. Insertion of the HIS3 gene halfway into the RPA49 coding region results in synthesis of a truncated A49 subunit that is incorporated into the polymerase. The truncated and wild-type subunits compete equally for assembly in the heterozygous diploid, although the wild type is phenotypically dominant.The yeast Saccharomyces cerevisiae possesses three nuclear RNA polymerases-A, B, and C (I, II, and III). These three forms of RNA polymerase can be distinguished by their subcellular localization, chromatographic behavior, subunit composition, sensitivity to a-amanitin, and promoter/ template specificity. Recent advances in the molecular genetics of yeast have made possible the functional dissection of the polymerases through cloning and sequencing of the genes encoding subunits and through selection of corresponding mutants (see refs. 1 and 2 for recent reviews).The single (essential) function of RNA polymerase A (pol A) is to transcribe the >100 ribosomal DNA units into the 35S ribosomal precursor (3), which is subsequently matured into the functional 18S, 5.8S, and 25S species (4). These RNA species together comprise 85% oftotal cellular RNA (5). Like the two other nuclear enzymes, yeast pol A has a complex subunit structure that includes a core of four subunits (A190, A135, AC40, and AC19) related to the 1,3'a2 eubacterial core enzyme (6) and a set of five small subunits (ABC27, ABC23, ABC14.5, ABC1Oa, and ABC1OB), which are common to all three yeast nuclear RNA polymerases (7). The corresponding genes have been cloned, sequenced, and found to encode essential components of the enzyme as judged from the properties of null mutants (6,(8)(9)(10)(11)(12). In addition, five other polypeptides (A49, A43, A34.5, A14, and A12.2) are associated with yeast pol A and are thus likely to be specific subunits of that enzyme (13,14).The gene encoding the 49-kDa subunit (A49) is ofparticular interest since this protein has been extensively studied at the biochemical level. Along with the subunit A34.5, A49 is easily dissociated from the rest of pol A, producing the form A*, which shows impaired transcriptional activity and increased sensitivity to a-amanitin as compared to the complete polymerase (14). In addition, the A49 subunit has been observed to copurify with an RNase H activity (15, 16), although the possibility of a contaminating enzyme with RNase H activity that copurifies wi...

Ca2ϩ signaling is important in many fundamental neuronal processes including neurotransmission, synaptic plasticity, neuronal development, and gene expression. In cerebellar Purkinje neurons, Ca 2ϩ signaling has been studied primarily in the dendritic region where increases in local Ca 2ϩ have been shown to occur with both synaptic events and spontaneous electrical activity involving P-type voltage-gated Ca 2ϩ channels (VGCCs), the predominant VGCC expressed by Purkinje neurons. Here we show that Ca 2ϩ signaling is also a prominent feature of immature Purkinje neurons at developmental stages that precede expression of dendritic structure and involves L-type rather than P-type VGCCs. Immature Purkinje neurons acutely dissociated from postnatal day 4-7 rat pups exhibit spontaneous cytoplasmic Ca 2ϩ oscillations. The Ca 2ϩ oscillations require entry of extracellular Ca 2ϩ , are blocked by tetrodotoxin, are communicated to the nucleus, and correlate closely with patterns of endogenously generated spontaneous and evoked electrical activity recorded in the neurons. Immunocytochemistry showed that L-, N-, and P/Q-types of VGCCs are present on the somata of the Purkinje neurons at this age. However, only the L-type VGCC antagonist nimodipine effectively antagonized the Ca 2ϩ oscillations; inhibitors of P/Q and N-type VGCCs were relatively ineffective.

Dihydroorotase, the third enzymatic activity of the pyrimidine pathway, is encoded in Saccharomyces cerevisiae by a single gene URA4, which is induced at the transcriptional level by accumulation of ureidosuccinic acid. A regulatory gene PPR2 (pyrimidine pathway regulatory 2) acting specifically on this step, has been characterized, cloned and sequenced. The main open reading frame is 384 nucleotides long and potentially codes for a basic protein, favoring a molecular mechanism involving direct binding of a regulatory protein to DNA. The short length of the PPR2 polypeptide chain and the presence of seven cysteine residues suggest that the active form of the protein is an oligomer assembled through disulphide bonds. An uninducible allele has been cloned and sequenced. The mutation corresponds to an A leads to T transversion changing a lysine triplet into an ochre codon. The uninducible phenotype of this mutant is completely suppressed by an ochre suppressor, strengthening the hypothesis that PPR2 acts on URA4 transcription through the synthesis of a regulatory protein.

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We selected a 5-fluorouracil-resistant, thermosensitive mutant of the uridine monophosphokinase step in Saccharomyces cerevisiae. The mutant displays very weak thermolabile uridine monophosphokinase activity and wild-type uridine diphosphokinase activity. Growth of the mutant at the non-permissive temperature causes immediate reduction of pyrimidine triphosphate pools to 10% of the wild-type level as well as significantly lowering total RNA and protein synthesis. These conditions also provoke derepression of the first gene of the pathway, URA2, at both the levels of enzymatic activity and transcription. The mutation segregates independently of all known genes of the pyrimidine biosynthetic pathway. The corresponding gene has been isolated on a 4.8 kb fragment by complementation of the mutant phenotype. The new gene, named URA6, codes for a 2.2 kb polyadenylated messenger RNA, exists in a single copy per haploid genome, and was mapped to the centromere of chromosome XI.

The expression of the metabotropic glutamate receptor mGluR1 was studied with Northern and Western blot analysis, with immunocytochemistry, and with Ca2+ digital imaging in the developing rat hypothalamus. mGluR1 is coupled to a G protein and activation by glutamate and related agonists leads to intracellular phosphotidylinositol hydrolysis and Ca2+ mobilization. mGluR1 RNA could be detected in embryonic hypothalamus, and by the day of birth and prior to the primary period of synaptogenesis, both mGluR1 RNA and protein were strongly expressed. In parallel experiments with digital imaging of cultured hypothalamic cells, some embryonic day 18 hypothalamic neurons and many astrocytes after 3 d in vitro showed Ca2+ responses to quisqualate and t-ACPD, and to glutamate in the absence of extracellular Ca2+. A greater number of embryonic neurons responded to NMDA than to agonists of the metabotropic receptor. With increased development time in culture, the number of neurons that responded to metabotropic glutamate receptor agonists increased. In the adult hypothalamus, mGluR1-immunoreactive neurons were widespread, and particularly dense in the dorsomedial, lateral, and anterior hypothalamus/preoptic areas, and in the mammillary body. Strongly immunoreactive cells were interspersed among neurons with no immunoreactivity. In developing neurons a diffuse immunostaining appeared along dendrites and somata. With time, beginning in the first week after birth, strongly stained puncta appeared, possibly associated with synaptic specializations. These puncta were numerous on dendrites of some adult neurons, and were the most strongly stained regions of neurons. Neurons developing in vitro at low neuron densities showed a development of mGluR1 immunoreactivity similar to that of neurons in vivo, but with a delayed progression of immunostaining. We found no obvious staining of axons or of astrocytes. A strong expression of mGluR1 protein was found in the hypothalamus during the first 2 postnatal weeks; this expression was partially reduced in adults. In contrast, cerebellum showed no reduction in mGluR1 protein in adults. Together these data suggest a complex regulation of mGluR1 during development, with sufficient expression of functional receptors in the developing hypothalamus to modulate morphogenesis and synaptogenesis, and later to play a role in transduction of glutamate signals in the adult. Different regions of the brain showed dramatic differences in the way each expresses mGluR1 during development.

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