Synapses onto different morphological types of retinal ganglion cells.

The percentage of bipolar and amacrine synapses onto ganglion cell dendrites of the ground squirrel has been determined by electron microscopy of cells impregnated by the Golgi method. One group of ganglion cells has mainly amacrine input (approximately 97 percent); the other group has an approximately equal bipolar and amacrine input. Morphologically distinct types of ganglion cells usually have a consistent synaptic input, but exceptions may exist.

Subsynaptic plate perforations: changes with age and experience in the rat.

The relative frequency of appearance of discontinuities in the postsynaptic thickening, or perforations in the subsynaptic plate, increased with age and experience. Rats reared from weaning in complex or social environments had a significantly higher proportion of occipital cortical synapses with perforations than did rats reared in isolation. In addition, the relative frequency of these perforations more than tripled between 10 and 60 days of age. Shifts in the frequency of perforations can occur independently of changes in the size of synpases. This result suggests a new potential mechanism of synaptic plasticity.

Galileo ultraviolet spectrometer experiment: initial venus and interplanetary cruise results.

The Galileo Extreme Ultraviolet Spectrometer obtained a spectrum of Venus atmospheric emissions in the 55.0- to 125.0-nanometer (nm) wavelength region. Emissions of helium (58.4 nm), ionized atomic oxygen (83.4 nm), and atomic hydrogen (121.6 nm), as well as a blended spectral feature of atomic hydrogen (Lyman-beta) and atomic oxygen (102.5 nm), were observed at 3.5-nm resolution. During the Galileo spacecraft cruise from Venus to Earth, Lyman-alpha emission from solar system atomic hydrogen (121.6 nm) was measured. The dominant source of the Lyman-alpha emission is atomic hydrogen from the interstellar medium. A model of Galileo observations at solar maximum indicates a decrease in the solar Lyman-alpha flux near the solar poles. A strong day-to-day variation also occurs with the 27-day periodicity of the rotation of the sun.

Differential repression of GAL4 and adjacent transcription activators by operators in the yeast GAL upstream activating sequence.

The upstream activating sequence of the adjacent and divergently transcribed GAL1 and GAL10 genes of Saccharomyces cerevisiae (UASG) contains at least three distinct classes of overlapping transcriptional control sites. The transcription activator GAL4 binds to four related sites in UASG and induces expression of GAL1 and GAL10 when galactose is available. We showed that UASG contains two additional positive control sites, designated GAL4/galactose-independent activating elements (GAEs), which reside at positions adjacent to or overlapping the GAL4-binding sites. When separated from neighboring sequences in UASG, the GAEs activate transcription independently of GAL4 with no requirement for galactose. In the intact GAL1-GAL10 divergent promoter region, their activity is ordinarily repressed by multiple negative control elements, the GAL operators. When galactose is available, GAL4 overcomes the activity of the GAL operators, while the putative GAE-binding proteins stay repressed. Combined, these results imply that distinct activators (GAL4 and GAE proteins) bound at adjacent or overlapping sites in UASG are differentially regulated by putative repressor proteins simultaneously bound at adjacent GAL operators. We surmise that GAE1 and GAE2 may have a physiological function other than regulation of galactose catabolism per se and discuss three hypotheses to account for their presence in UASG.

Saccharomyces cerevisiae GAL1-GAL10 divergent promoter region: location and function of the upstream activating sequence UASG.

The GAL1 and GAL10 genes, separated by 680 base pairs and divergently transcribed on chromosome 2 of Saccharomyces cerevisiae, were separately fused to the lacZ gene of Escherichia coli so that beta-galactosidase synthesis in S. cerevisiae reflected GAL1 and GAL10 promoter function. Analysis of two sets of deletions defined a 75-base-pair sequence, located ca. midway between the transcription initiation regions of GAL1 and GAL10, that mediates GAL4-dependent induction of both genes. Deletion of various parts of this sequence (called the GAL upstream activating sequence or UASG) reduced GAL1 and GAL10 induction about equally. Sequences in the GAL10-proximal half of UASG in some sequence contexts functioned independently of sequences in the GAL1-proximal half of UASG. A 33-base-pair deletion of the GAL10-proximal half of UASG drastically reduced induction. Deletions between UASG and the GAL1 TATA box caused beta-galactosidase to be synthesized at an unexpectedly high basal level, that is, in the absence of galactose and GAL4 product. Some of these mutations also reduced the repression caused by glucose.

Opposing regulatory functions of positive and negative elements in UASG control transcription of the yeast GAL genes.

The yeast GAL1 and GAL10 genes are transcribed at a remarkably low basal level when galactose is unavailable and are induced by over 4 orders of magnitude when it becomes available. Approximately six negative control elements (designated GAL operators GALO1 to GALO6) are located adjacent to or overlapping four binding sites for the transcription activator GAL4 in the GAL upstream activating sequence UASG. The negative control elements contribute to the broad range of inducibility of GAL1 and GAL10 by inhibiting two GAL4/galactose-independent activating elements (GAE1 and GAE2) in UASG. In turn, multiple GAL4-binding sites in UASG are necessary for GAL4 to overcome repression by the negative control elements under fully inducing conditions. When glucose in addition to galactose is available (repressing conditions), the ability of GAL4 to activate transcription is diminished as a result of its reduced affinity for DNA and the reduced availability of inducer. Under these conditions, the negative control elements inhibit transcriptional activation from the glucose-attenuated GAL4 sites, thus accounting at least in part for glucose repression acting in cis. A normal part of transcriptional regulation of the GAL1 and GAL10 genes, therefore, appears to involve a balance between the opposing functions of positive and negative control elements.

TSF3, a global regulatory protein that silences transcription of yeast GAL genes, also mediates repression by alpha 2 repressor and is identical to SIN4.

TSF3 encodes one of six (TSF1 to TSF6) recently identified global negative regulators of transcription in Saccharomyces cerevisiae. Mutant tsf3 strains exhibit defects in transcriptional silencing of the GAL1 promoter, allow expression from upstream activation sequence-less promoters, and exhibit pleiotropic defects in cell growth and development. Here we show that TSF3 is involved in transcriptional silencing mediated by the alpha 2 repressor and demonstrate that specific systems of transcriptional silencing may depend on the more global role of TSF3. Cloning and sequencing of TSF3 allowed us to predict a 974-amino-acid gene product identical to SIN4, a negative regulator of transcription of the HO (homothallism) mating type switching endonuclease. TSF3 disruptions are not lethal but result in phenotypes similar to those of the originally isolated alleles. Our results, together with those of Y. W. Jiang and D. J. Stillman (Mol. Cell. Biol. 12:4503-4514, 1992), suggest that TSF3 (SIN4) affects the function of the basal transcription apparatus, and this effect in turn alters the manner in which the latter responds to upstream regulatory proteins.

TSF1 to TSF6, required for silencing the Saccharomyces cerevisiae GAL genes, are global regulatory genes.

The Saccharomyces cerevisiae GAL1 and GAL10 genes are controlled in response to the availability of galactose and glucose by multiple activating and repressing proteins bound at adjacent or overlapping sites in UASG. Negative control elements in UASG, designated GAL operators GALO1 to GALO6, are required to silence basal level transcription of GAL1 and GAL10 when galactose is absent. We isolated and characterized recessive mutations in six nuclear genes, TSF1 to TSF6, that impair silencing of GAL1 and GAL10 gene expression. Surprisingly, the results of several experiments suggest that the TSF genes encode global regulatory factors. tsf1 to tsf6 mutations derepressed expression from yeast CYC-GAL hybrid promoters (fused to lacZ) that harbor a variety of operator sequences, and caused pleiotropic defects in cell growth, mating, and sporulation. S1 mapping and Northern blot results for tsf3 suggest that the molecular defect is at the transcriptional level. Mutant phenotypes were additive in certain combinations of tsf double mutants, implying that more than one silencing pathway is involved in TSF1 to TSF6 function. Most significantly, mutations in all six TSF1 to TSF6 genes activated expression from GAL1 and CYC1 promoters (fused to lacZ) lacking upstream activating sequences. Combined, the simplest interpretation of these results is that TSF1 to TSF6 encode factors that control the function of the basic RNA polymerase II transcriptional machinery.

Construction and characterization of E. coli promoter-probe plasmid vectors. III. pBR322 derivatives with deletions in the tetracycline resistance promoter region.

Deletions of the promoter region for the tetracycline-resistance (Tcr) gene(s) of pBR322 were constructed in order to generate new promoter-probe plasmid cloning vectors. The deletions were constructed in vitro by exonuclease digestion at the HindIII site and blunt-end ligation of the digestion products. Plasmids which lost the HindIII site but retained the EcoRI site carried deletions ranging from 5 to 60 bp. Some of the plasmids lacked the nucleotide sequences required for initiation of transcription from the Tcr promoter and "anti-Tcr" promoter. Three of the promoter-deletion plasmids (containing deletions of 5-29 bp) formed tight-binding complexes with RNA polymerase in vitro, despite their tetracycline sensitive phenotype. One deletion plasmid, pPV33, retained three out-of-phase stop codons located between the promoter-cloning site (EcoRI) and the translational start codon for the tetracycline resistance gene. These features give pPV33 several advantages over previously described promoter-cloning vehicles.

Construction and characterization of E. coli promoter-probe plasmid vectors. II. RNA polymerase binding studies on antibiotic-resistance promoters.

The binding of Escherichia coli RNA polymerase to antibiotic-resistance promoters was examined using the nitrocellulose filter assay. Four filter-retainable HaeIII fragments were observed with pBR322 and the promoter-probe plasmids, pBRH1, pBRH2 and pBRH4. Of the three fragments studied, two were shown to carry promoters for the ampicillin (Ap) and tetracycline (Tc) resistance genes, while the third present in pBRH1 appears to be the promoter for colicin E1 immunity (Colimm). Although the formation of filter-retainable complexes involving the Tcr promoter was sensitive to high salt, Apr promoter complexes were not. It was also shown that plasmids containing only the "firm-binding" portion of the Tcr promoter could still bind RNA polymerase in vitro despite the fact that these plasmids confer no in vivo Tcr. Additional filter-binding experiments performed with AluI-digested pBR322 DNA revealed the presence of a fifth RNA polymerase binding site on pBR322. This site is probably the promoter for the 100 bp transcript thought to be involved in the initiation of plasmid replication. An analysis of the recombinant plasmid (pKTR25) which carries the Kan-B portion of the EcoRI kanamycin (Kn) resistance fragment revealed that this fragment contains two RNA polymerase binding sites. We believe that these sites are responsible for the insertional activation of the Tcr gene and may be the promoters for the Knr and fusidic acid (Fa) resistance genes.

Construction and characterization of E. coli promoter-probe plasmid vectors. I. Cloning of promoter-containing DNA fragments.

Derivatives of the Escherichia coli drug-resistance plasmid pBR316 have been constructed which act as molecular probes for promoter-containing DNA restriction fragments from various prokaryotic genomes. The plasmids, designated pBRH1 and pBRH3B, contain a unique EcoRI restriction site located within the promoter for the tetracycline resistance (Tcr) gene. This site was created by the insertion of a chemically synthesized octanucleotide, containing the EcoRI cleavage sequence, into the HindIII site of pBR316. Base-pair alterations within the Tc promoter produced by this insertion resulted in a substantial reduction (pBRH3B) or elimination (pBRH1) in ability of these plasmids to confer Tc resistance to the host strain. Cloning of EcoRI-cleaved foreign DNA fragments into the EcoRI site of these plasmids allows for the isolation of recombinant transformants with Tcr levels greater than that of the plasmid vector. Further characterization of these recombinant plasmids demonstrates that the Tcr phenotype is dependent upon the orientation of the inserted fragment, but not on the molecular weight. We have concluded that these fragments carry promoters which, in the proper orientation, allow for the transcription of the Tcr gene. The utility of these "promoter-probe" plasmids lies in the ability to select for promoter-containing DNA fragments by insertional activation of the Tcr gene.

Sequence of the Saccharomyces cerevisiae YTP1 gene encoding a deduced novel type-III integral membrane protein with domains of sequence similarity to mitochondrial electron-transport enzymes.

The nucleotide sequence is reported for the Saccharomyces cerevisiae YTP1 (yeast putative transmembrane (TM) protein) gene, encoding a novel deduced protein of 459 amino acids (aa) in length (51 643 Da). The Ytp1 protein appears by computer analysis (hydropathy plots in conjunction with the combined predictions of several Internet on-line programs that deduce protein structure from primary sequence data) to be a type-III integral TM protein containing 10 or 11 TM-spanning domains. Blocks of aa sequence similarity, predominantly to mitochondrial electron transport proteins, are consistent with the notion that Ytp1 is an integral TM protein and may reflect some aspect of its functional role. The C terminus of Ytp1 is both hydrophilic and highly negatively charged, with 11 of the last 33 aa corresponding to Glu or Asp. Although Northern blot analysis indicates that this gene is expressed, a disruption of YTP1 shows that it is not essential. YTP1 is located between SIN4 (TSF3) and KEX2 (SRB1) at position 205 (kb) on the chromosome XIV physical map.

RLR1 (THO2), required for expressing lacZ fusions in yeast, is conserved from yeast to humans and is a suppressor of SIN4.

We isolated a mutation (rlr1-1; required for lacZ RNA) in the Saccharomyces cerevisiae (Sc) RLR1 gene as a suppressor of sin4, a component of the Mediator subcomplex of the RNA polymerase II holoenzyme and a determinant of chromatin structure. RLR1 encodes a deduced protein found also in fission yeast, nematode worms, and humans. The presence of these orthologs suggests that Rlr1 family members comprise a class of putative KEKE motif-containing proteins, characteristic of certain chaperones as well as regulators and subunits of the mammalian 20S proteasome. A role for RLR1 (THO2) in transcription appears to occur at a step subsequent to transcription initiation (see also Piruat, J.I. and Aguilera, A., 1998. EMBO J. 17, 4859-4872); Sc genes fused to the reporter gene lacZ were expressed at a very low level, while the corresponding native chromosomal genes were expressed at approximately normal levels in rlr1 mutants. Our studies show that rlr1 mutations cause a wide range of growth defects in addition to their novel affect on lacZ.

Light and electron microscopy of the ground squirrel retina: functional considerations.

Light and electron microscopy of Golgi-impregnated ground squirrel retinas have revealed a range of morphological subtypes of bipolar, amacrine, and ganglion cells. There are at least seven subtypes of bipolar cells. Those subtypes in which the somata were high (sclerad) in the inner nuclear layer (3 subtypes) had axon terminals low (vitread) in the inner plexiform layer, and those with somata low in the inner nuclear layer (4 subtypes) had axon terminals high in the inner plexiform layer. The bipolar subtypes with high axon terminals made flat contacts with receptor cells, whereas all but one of the bipolar subtypes with low axon terminals made ribbon-related contacts with receptor cells. There are at least five subtypes of amacrine cells. The two subtypes which the Golgi method revealed most frequently were a broad-field, unistratified neuron with a dendritic spread in excess of 1,000 mum and a narrow-field, diffuse neuron with a dendritic spread of about 30 mum. The broad-field, unistratified cell had the lowest proportion of amacrine vs. bipolar cell synaptic input of the amacrine subtypes (43%), whereas the narrow-field, diffuse cell had one of the greatest proportions of amacrine cell input (96%). There are at least 15 subtypes of ganglion cells. The proportion of synaptic inputs to these cells ranged from 21% to 100% amacrine cell synapses. An attempt has been made to relate this new knowledge of retinal circuitry to the physiological output of the ganglion cells.

Anatomical evidence for cone and rod-like receptors in the gray squirrel, ground squirrel, and prairie dog retinas.

In the gray squirrel (Sciurus carolinensis), the prairie dog (Cynomys ludovicianus), and the Mexican and 13-line ground squirrels (Citellus mexicanus and C. tridecemlineatus) there exist two distinct classes of photo-receptors that have cone-like and rod-like anatomical features respectively. These two receptor classes were previously known to exist in the gray squirrel, but only the cone-like (C) receptor had been observed in the other species. We have now found small numbers of rod-like (R) receptors in the other species as well. R-receptors comprise about 40% of the receptors in the gray squirrel, 10% of the receptors in the prairie dog, and 4-5% of the receptors in the two species of ground squirrel. This paper describes certain light and electron microscopic features of these two receptor classes including their synaptic connections with second-order cells and with each other. We find that the C-receptor has a morphology and synaptic organization characteristic of other mammalian cones. However, the R-receptor differs from other mammalian rods in certain morphological respects, and its synaptic organization has both cone and rod characteristics as well as some unusual features.

A re-examination of anatomical plasticity in the rat retina.

Previous investigators have reported an increase in numbers of amacrine synapses in the inner plexiform layer (IPL) of the rat retina following light deprivation, and an increase in amacrine along with a decrease in bipolar synapses following light damage. Since there are several points of disagreement between the published reports on this subject we undertook a more detailed study of the effects of light deprivation and light damage on the retina. Four groups of eight male albino rat pups (14 days old) were raised for eight weeks under different conditions: (1) unsutured, bright light reared (UB); (2) bilaterally lid-sutured, bright light reared (SB); (3) unsutured, low light reared (UL); and (4) bilaterally lid-sutured, dark reared (SD). The intensity of the light given the UL group was equated with that striking the corneas of the SB group. Light microscopy showed that the retinas of the SB as well as the UB groups had almost complete degeneration of the outer retinal layers, indicating that even low intensity light, when continuous, causes severe retinal damage. The SD group was thicker in many of the retinal layers compared to the UL (control) group. Electron microscopy revealed that there were no significant changes in the incidences of any type of synapse in the IPL following light deprivation or light damage when averaged over total depth. This is in contradiction to the reports of other investigators. However, when the IPL was analyzed by levels, the incidence of amacrine-ganglion synapses was signficantly greater (p less than 0.05) in groups UB and SD, but only in the outer third of the IPL. Thus, extensive postnatal plasticity of IPL synapses in the rat retina did not occur under our experimental conditions. We found, at best, only limited effects which were confined to the amacrine-ganglion synapses.

Saccharin effects on the urinary physiology and urothelium of the rat when administered in diet or drinking water.

Three groups of 4 rats each were given 5% saccharin in the diet, 4% saccharin in the drinking water, or control diet, and were observed for changes in urinary physiology and pathology. Rats receiving saccharin in the water showed a significantly increased urinary osmolality over control values. Rats receiving saccharin in the diet showed significant increases in food and water consumption, urine elimination, and urinary sediment. Only the latter rats exhibited histopathological changes consisting of two instances of mild hyperplasia and one instance of mild cellular vacuolization.

The role of urinary physiological changes in the genesis of urothelial lesions in mice given 4-ethylsulfonylnaphthalene-1-sulfonamide, acetazolamide, and oxamide.

BALB/c female mice were administered several compounds, including 4-ethylsulfonylnaphthalene-1-sulfonamide, acetazolamide, and oxamide, in the diet for six weeks. Fresh urine samples were analyzed three times per week for pH, osmolality, micro-crystals, and protein; and a histopathological evaluation was made of the urothelium at the end of the six weeks test. Incidences of hyperplasia, nodular hyperplasia, vacuolization, ulceration and acute inflammation of the bladder urothelium appeared to be related to the osmolality of the urine and the micro-crystalluria experienced by the mice. Correlation coefficients between lesions and urinary osmolality or crystals were -0.69 (p less than 0.0001) and 0.31 (p less than 0.03), respectively, at the 5% significance level.

Urothelial lesions in mice given 4-ethylsulfonylnaphthalene-1-sulfonamide, acetazolamide, and oxamide.

4-Ethylsulfonylnaphthalene-1-sulfonamide, acetazolamide and oxamide were administered in the diet to female BALB/c mice for varying periods of time from three to eighty weeks. All three compounds induced lesions in the urothelium. In the bladder, these included simple hyperplasia, nodular hyperplasia, inflammation and calculi. Similar lesions were observed in the ureter and urethra, along with a novel lesion, diverticulum, in the ureter. The diverticular lesions existed as down-growths of the transitional epithelium which often extended from the mucosa through the muscle layers to the adventitial surface. The etiology of the lesions appeared to be related to urinary physiological alterations (crystalluria, calculi, hypoosmolality) caused by administration of the compounds.

In vitro transformation potential of N-polycyclic aromatic hydrocarbons in rat tracheal epithelial cells.

Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are environmental contaminants and potential human airway carcinogens. Agents of this class show a wide range of potencies for toxicity, mutagenicity and carcinogenicity that are associated with the structure of the PAH and the position of the nitro group. In order to evaluate the effect of nitro substitution on in vitro biological activity, the cytotoxicity and transformation potential of two parent PAHs, pyrene and chrysene, and a series of nitro derivatives were examined in the rat tracheal epithelial (RTE) cell system. The nitro derivatives, but not pyrene or chrysene, produced dose-dependent decreases in the colony forming efficiency of the RTE cells. The most cytotoxic agents were 1,6-dinitropyrene and 6-nitrochrysene with ED(50)s of 1.6 mum and 5.9 mum, respectively, followed by 4-nitropyrene and 1-nitropyrene with ED(50)s of 26.3 mum and 44.5 mum, respectively. These compounds were evaluated for transformation potential at three treatment levels that spanned the cytotoxic range, and the assays were scored for morphologically transformed preneoplastic colonies. The control or spontaneous transformation frequency in this series of experiments was 1.79 +/- 0.47 (x 10(-4)). 6-Nitrochrysene and 1,6-dinitropyrene were the only compounds that produced transformation frequencies (12.17 x 10(-4) and 9.68 x 10(-4), respectively) that were statistically different from control. The maximum transformation frequencies of the compounds were compared with published data for liver tumorigenicity in the newborn mouse assay. The orders for tumorigenicity and transformation were the same (1,6-dinitropyrene > 4-nitropyrene > 1-nitropyrene approximately - pyrene and 6-nitrochrysene > chrysene), and the relative potencies of the compounds were similar in the two assays. These results suggest that RTE cells are capable of metabolizing nitro-PAHs to reactive products, and that, within this limited class of compounds, in vitro transformation data in the RTE cell system may be correlated with tumorigenicity in animal studies.