L-Type calcium channels mediate calcium oscillations in early postnatal Purkinje neurons.

Ca(2+) signaling is important in many fundamental neuronal processes including neurotransmission, synaptic plasticity, neuronal development, and gene expression. In cerebellar Purkinje neurons, Ca(2+) signaling has been studied primarily in the dendritic region where increases in local Ca(2+) have been shown to occur with both synaptic events and spontaneous electrical activity involving P-type voltage-gated Ca(2+) channels (VGCCs), the predominant VGCC expressed by Purkinje neurons. Here we show that Ca(2+) 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(2+) oscillations. The Ca(2+) oscillations require entry of extracellular Ca(2+), 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(2+) oscillations; inhibitors of P/Q and N-type VGCCs were relatively ineffective. Release of Ca(2+) from intracellular Ca(2+) stores significantly amplified the Ca(2+) signals of external origin. These results show that a somatic signaling pathway that generates intracellular Ca(2+) oscillations and involves L-type VGCCs and intracellular Ca(2+) stores plays a prominent role in the Ca(2+) dynamics of early developing Purkinje neurons and may play an important role in communicating developmental cues to the nucleus.

Dynamic behavior of the ends of growing parallel fibers in early postnatal rat cerebellum.

The molecular layer of the cerebellum contains parallel fibers, the axons of granule neurons. We have examined the morphology and behavior of parallel fiber growth cones in the early postnatal rat cerebellum using the fluorescent tracer DiI. Parallel fiber growth cones distributed into three categories based on size and shape: short torpedo-like, long torpedo-like, and lamellopodial in form. The torpedo-like growth cones were modified by the addition of lamellopodia and/or filopodia, and the lamellopodial growth cones were often decorated with a filopodium. These three different growth cone morphologies were found throughout the growing region of the molecular layer. The nascent axons elaborated by premigratory granule neurons differed form the longer axons of more developed neurons in that they often had forked growth cones and extensive lamellopodial decoration along the axon shaft. Growth cones in living slices closely resembled those observed in the fixed preparations. The living growth cones exhibited frequent lamellopodial rearrangement and a side-to-side headwaving movement. The axon proximal to the growth cone was also dynamic. The axons curved and undulated, and mobile swellings formed along the axon shaft. These observations show that the growth cones of parallel fibers are similar to growth cones described for axons in other developing systems in terms of size, morphological characteristics, and dynamic behavior.

Growth cone interactions with purified cell and substrate adhesion molecules visualized by interference reflection microscopy.

The migration of growth cones on substrates consisting of naturally occurring cell adhesion molecules has been extensively studied in cell culture. However, relatively little is known about how growth cones contact the substrate or how the patterns of contact change as growth cones move forward. We have examined the interactions of chick retinal ganglion cell growth cones with laminin, merosin, N-cadherin, L1 and poly-L-lysine by time-lapse interference reflection microscopy (IRM) using a laser scanning confocal microscope. In images obtained by IRM, areas of a cell that are closely apposed to the substrate appear dark whereas areas that are farther away appear light. Growth cones on Jaminin and merosin were almost uniformly light, indicating that very little of the membrane was in close contact with the substrate. Growth cones on N-cadherin had a mottled appearance with some relatively large dark gray areas. The proximal portions of filopodia often were dark, in contrast to those on laminin and merosin which were light. In addition, growth cones on N-cadherin had numerous dark gray punctate regions of close association with the substrate. Growth cones on L1 had darker regions than growth cones on other substrates and these comprised a larger fraction of their area. There also were differences in the temporal dynamics of growth cone interactions with different substrates and these differences correlated with differences in rates of growth. None of the contacts observed in growth cones were as dark or stable as focal contacts of fibroblasts.

Expression of the neural axon adhesion molecule L1 in the developing and adult rat brain.

L1 is a developmentally regulated adhesion molecule that may play a role in some aspects of axonal guidance. With Northern blots we find peak expression of L1 RNA at postnatal day 1 (P1) in the developing rat brain. Western blots show a peak of protein on P15. The major form of L1 is 200 kDa, but lower molecular mass forms are found including 140 and 80 kDa, representing, respectively, the extracellular and intracellular regions of L1. All molecular mass forms of L1 change during development. Although expressed at lower levels than during development, L1 is found in all brain regions in the adult rat. Different regions of the brain show differential expression and regulation of L1 and its peptide fragments. For instance, the hypothalamus showed an enhanced L1-80/L1-200 ratio at P10 and P15 relative to that expressed by cerebellum and hippocampus. Cerebellar granule cells in culture showed strong L1-200 and almost no L1-60, -80, or -140, in contrast to the intact cerebellum at the same age, which showed weaker L1-200 and strong L1-60, -80, and -140. Control experiments indicated that the L1 proteolytic cleavage found in different developing brain regions occurred in vivo and was not a result of sample preparation. The amount of L1-200 in cultured granule cells was proportional to the measured length of the growing axon. Neuronal activity (increased with 25 mM K+, 100 microns N-methyl-D-aspartate, and 100 microns 4-aminopyridine) enhanced L1 transcription and translation. Together, these data suggest differential regulation of L1 expression and proteolytic cleavage specific for developmental ages and brain regions.

Developmental regulation of the hypothalamic metabotropic glutamate receptor mGluR1.

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.

Altered promoter binding of the TATA box-binding factor induced by the transcriptional activation domain of VP16 and suppressed by TFIIA.

The acidic transcriptional activation domain of the Herpes simplex virus protein VP16 has been shown to bind directly to both the TATA box-binding factor TBP and the general initiation factor TFIIB. Using DNase I footprinting assays, we have shown here that the VP16 activation domain qualitatively alters binding of Saccharomyces cerevisiae TBP to a TATA sequence in DNA. The effect of VP16 on promoter binding by TBP was reduced by mutations in VP16 known to reduce transactivation and could not be overcome by increasing the amount of TBP used in the footprinting assays. However, the association of yeast TFIIA with TBP on the promoter reversed the VP16-mediated effect and restored normal binding of TBP to the promoter. We suggest that VP16 induces a conformational change in TBP which alters its binding to promoter DNA, and that this effect of VP16 is suppressed by TFIIA.

Characterization and mutagenesis of the gene encoding the A49 subunit of RNA polymerase A in Saccharomyces cerevisiae.

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 douglasii subunits based on a polymorphism existing between the subunits of RNA 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.

Identification of three mammalian proteins that bind to the yeast TATA box protein TFIID.

The TATA box binding transcription factor TFIID of S. cerevisiae was used as a ligand for affinity chromatography. Polypeptides that bind specifically to yeast TFIID (TFIID-associated proteins, DAPs) were purified from human HeLa (heDAPs) and calf thymus (ctDAPs) whole cell extracts. Both heDAP and ctDAP fractions altered the binding of TFIID to the TATA element, and substituted for the TFIIA transcription activity in a reconstituted in vitro system. The heDAP fraction also behaved like TFIIA in its ability to form a promoter-TFIID-TFIIA complex and to recruit TFIIB to such a complex. The interaction of DAPs with TFIID can confer heat-resistance (47 degrees C) on recombinant yeast or human TFIID. SDS-PAGE analysis revealed that three polypeptides from HeLa extracts specifically bound to yTFIID columns (heDAP35, heDAP21, and heDAP12). These data suggest that a multi-subunit transcription factor with the properties of TFIIA can bind to TFIID in the absence of DNA.

Primary structure of the S. cerevisiae gene encoding uridine monophosphokinase.

In the yeast Saccharomyces cerevisiae, the biosynthesis of both pyrimidine nucleoside triphosphates UTP and CTP is dependent on the activity of the uridine monophosphokinase step. We have determined the sequence of the uridine monophosphokinase gene. The coding region is 615 base pairs long and encodes 205 amino acids (22,500 daltons). The 5' terminus is comprised of a 17 amino acid-long hydrophobic leader sequence which is not present in genes encoding adenylate kinases. The coding region shows a strong degree of homology with the cytosolic adenylate kinases of vertebrates, and a lesser degree of homology with yeast and E. coli adenylate kinases.

Genetic characterization and isolation of the Saccharomyces cerevisiae gene coding for uridine monophosphokinase.

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.

Yeast regulatory gene PPR1. II. Chromosomal localization, meiotic map, suppressibility, dominance/recessivity and dosage effect.

The Saccharomyces cerevisiae gene PPR1 encodes a positive regulator of the expression of the two unlinked structural genes URA1 and URA3. The gene has been mapped to a position 6.5 cM from the centromere of chromosome XII. Uninducible alleles have been selected and used to establish a meiotic map. Suppressible alleles have been identified. The sequencing of a suppressible allele confirms the nonsense nature of the mutation as well as the reading frame deduced from the nucleotide sequence. No evidence of intracistronic complementation was found, and enzymatic analysis of leaky mutants did not reveal any mutations dissociating regulation of URA1 from that of URA3. Three in vitro-constructed deletions of PPR1 have been integrated at the chromosomal locus, giving strains with a completely negative phenotype. These deletion mutants display the wild-type basal level of URA1 and URA3 expression and show a semi-dominant phenotype in heteroallelic ppr1+/ppr1-delta diploids. Amplifying PPR1 by introduction into yeast on a multicopy vector increases the induction factor of URA1 and URA3 expression. These results show that the extent of regulation of the two structural genes is dependent on the concentration of the active PPR1 protein.

Complete sequence of a eukaryotic regulatory gene.

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.