Quantification of iNOS mRNA with reverse transcription polymerase chain reaction directly from cell lysates.

Inducible nitric oxide synthase (iNOS) is a member of a family of primary inflammatory response genes. Quantitative measurement of iNOS mRNA levels is important for the study of gene expression of this enzyme during the process of inflammation. We report here a method for quantitative measurement of iNOS mRNA levels with rtPCR directly from cells lysed with a single step phenol/chloroform/ether extraction. Using a mouse macrophage cell line, J774.2, which expresses iNOS mRNA upon LPS + IFN-gamma treatment as the model, the effects of the extraction on iNOS mRNA recovery and cytosolic RNase removal have been studied. The cells are lysed and RNases denatured and removed by phenol/chloroform extraction. Trace amounts of the phenol partitioned in the samples are then removed by ether extraction. After the extraction, the samples can be used directly for reverse transcription and PCR without further purification of RNA. The recovery of specific mRNA is not affected by the extraction procedure and externally added iNOS cRNA shows no degradation by the extracted cell lysates. Measurement of iNOS mRNA with this procedure is linear using serially double-diluted cells in the range from 94 to 6000 cells. The efficiencies of rtPCR of iNOS wild-type and deletion cRNAs are also compared in our study. By controlling the molecular size of the deletion construct to within 10% of that of the wild type and maintaining PCR cycling below 25 cycles, the rtPCR efficiencies of iNOS wild type and deletion are identical. The detection of rtPCR products is enhanced by hybridization with specific probes. Under these conditions, iNOS mRNA concentration can directly be calculated from the internal standard in each tube without a standard curve. We conclude that our procedure provides an accurate method for quantitative measurement of iNOS mRNA from limited amount of cells without complete RNA isolation.

Mutational analysis of the Shab-encoded delayed rectifier K(+) channels in Drosophila.

K(+) currents in Drosophila muscles have been resolved into two voltage-activated currents (I(A) and I(K)) and two Ca(2+)-activated currents (I(CF) and I(CS)). Mutations that affect I(A) (Shaker) and I(CF) (slowpoke) have helped greatly in the analysis of these currents and their role in membrane excitability. Lack of mutations that specifically affect channels for the delayed rectifier current (I(K)) has made their genetic and functional identity difficult to elucidate. With the help of mutations in the Shab K(+) channel gene, we show that this gene encodes the delayed rectifier K(+) channels in Drosophila. Three mutant alleles with a temperature-sensitive paralytic phenotype were analyzed. Analysis of the ionic currents from mutant larval body wall muscles showed a specific effect on delayed rectifier K(+) current (I(K)). Two of the mutant alleles contain missense mutations, one in the amino-terminal region of the channel protein and the other in the pore region of the channel. The third allele contains two deletions in the amino-terminal region and is a null allele. These observations identity the channels that carry the delayed rectifier current and provide an in vivo physiological role for the Shab-encoded K(+) channels in Drosophila. The availability of mutations that affect I(K) opens up possibilities for studying I(K) and its role in larval muscle excitability.

Calcineurin subunit B is required for normal vegetative growth in Neurospora crassa.

A series of Neurospora crassa mutants affected in the ability to regulate entry into conidiation (an asexual developmental program) were isolated by using an insertional mutagenesis procedure followed by a screening protocol. One of the mutants isolated by this approach consisted entirely of cells with an abnormal morphology. The mutant produces chains of swollen septated cells. The developmentally regulated ccg-1 gene is constitutively expressed in these cells, suggesting that they have entered the conidial developmental program. The insertionally disrupted gene cnb-1 was isolated by plasmid rescue and found to encode calcineurin B, the regulatory subunit of the Ca2+ and calmodulin-dependent protein phosphatase calcineurin. The data demonstrate that calcineurin B is required for normal vegetative growth in N. crassa and suggest that the cnb-1 mutant is unable to repress entry into the asexual developmental program. The results suggest that Ca2+ may play an important role in regulating fungal morphology.

The isolation and characterization of nrc-1 and nrc-2, two genes encoding protein kinases that control growth and development in Neurospora crassa.

Using an insertional mutagenesis approach, a series of Neurospora crassa mutants affected in the ability to control entry into the conidiation developmental program were isolated. One such mutant, GTH16-T4, was found to lack normal vegetative hyphae and to undergo constitutive conidiation. The affected gene has been named nrc-1, for nonrepressible conidiation gene #1. The nrc-1 gene was cloned from the mutant genomic DNA by plasmid rescue, and was found to encode a protein closely related to the protein products of the Saccharomyces cerevisiae STE11 and Schizosaccharomyces pombe byr2 genes. Both of these genes encode MAPKK kinases that are necessary for sexual development in these organisms. We conclude the nrc-1 gene encodes a MAPKK kinase that functions to repress the onset of conidiation in N. crassa. A second mutant, GTH16-T17, was found to lack normal vegetative hyphae and to constitutively enter, but not complete, the conidiation program. The affected locus is referred to as nrc-2 (nonrepressible conidiation gene #2). The nrc-2 gene was cloned and found to encode a serine-threonine protein kinase. The kinase is closely related to the predicted protein products of the S. pombe kad5, and the S. cerevisiae YNRO47w and KIN82 genes, three genes that have been identified in genome sequencing projects. The N. crassa nrc-2 gene is the first member of this group of kinases for which a phenotype has been defined. We conclude a functional nrc-2-encoded serine/threonine kinase is required to repress entry into the conidiation program.

The isolation and characterization of a Neurospora crassa gene (ubi::crp-6) encoding a ubiquitin-40S ribosomal protein fusion protein.

We have isolated and sequenced a Neurospora crassa gene encoding a single copy of ubiquitin (UBI) fused to the S27a ribosomal (r) protein. We have opted to name this gene the ubiquitin/cytoplasmic r-protein gene 6 (ubi::crp-6). The ubi::crp-6 gene generates a 700-nucleotide (nt) transcript. It shares a 700-bp regulatory region with the cytoplasmic r-protein gene 5 (crp-5), a gene encoding the N. crassa S26 r-protein. The two genes are transcribed divergently from the common regulatory region and their mRNA levels are regulated in parallel during growth on a variety of carbon sources.

A cis-acting region required for the regulated expression of grg-1, a Neurospora glucose-repressible gene. Two regulatory sites (CRE and NRS) are required to repress grg-1 expression.

Grg-1 is a Neurospora gene which was identified as being a highly expressed glucose-repressible gene. A cis-acting regulatory region required for the regulated expression of grg-1 has been characterized. The regulatory region is found between 440 and 500 nucleotides upstream of the first major grg-1 start of transcription site and contains two distinct cis-acting regulatory elements. The upstream element consists of the sequence GTGACGTCAC, which is identical to the previously identified CRE (Cyclic AMP-Responsive Element). The second element is a newly defined cis-acting regulatory site. The element has the sequence TTGCTAGCAA and has been named NRS (Neurospora Repressor Site). DNA binding proteins can be shown to bind to both of these cis-acting regulatory elements. Experiments in which these sites were deleted demonstrate that both cis-acting regulatory elements are required to turn off the in vitro expression of the grg-1 gene under conditions of glucose sufficiency.

Isolation, sequencing, and characterization of crp-5, a gene encoding a Neurospora ribosomal protein.

A Neurospora crassa cytoplasmic ribosomal protein gene, crp-5, has been isolated and characterized. The cDNA was isolated by a differential screening of a cDNA library for glucose-inducible genes. The cDNA was subsequently used to identify and isolate crp-5 genomic sequences. Computer analysis of the DNA sequences showed that they contain an open reading frame which encodes a protein homologous to the rat ribosomal protein S26. The crp-5 mRNA levels are regulated in a carbon-source-dependent manner. The organization of the gene and the region upstream of the coding sequences are discussed.

Developing Dictyostelium cells contain the lysosomal enzyme alpha-mannosidase in a secretory granule.

The prespore vesicle (PSV) is an organelle which secretes spore coat proteins and gal/galNAc polysaccharides from prespore cells of Dictyostelium. By combining the techniques of protein A-gold immunocytochemistry and ricin-gold affinity cytochemistry we have demonstrated colocalization of the lysosomal enzyme alpha-mannosidase with gal/galNAc polysaccharides in prespore vesicles and the spore coat. To determine the origin of prespore vesicles a series of pulse-chase experiments were performed. Cells were labeled with [35S]methionine or [35S]sulfate at different times during development and allowed to differentiate in the presence of unlabeled methionine or sulfate for various periods of time. The cells were homogenized and intracellular organelles were separated using Percoll density gradient centrifugation. The distribution of [35S]methionine-labeled alpha-mannosidase and [35S]sulfate-labeled glycoproteins in the Percoll gradients was determined. It was found that prespore vesicles contained protein which was previously found in lysosomes. Newly labeled protein also entered these vesicles. The data suggest that developing Dictyostelium cells either restructure preexisting lysosomes into prespore vesicles or transport protein between these two organelles. We propose that secretory granules and lysosomes may have a common biosynthetic origin and may be evolutionarily related.

Developing Dictyostelium discoideum cells contain two distinct acid hydrolase-containing vesicles.

Two distinct populations of acid hydrolase-containing vesicles have been found in developing Dictyostelium discoideum cells. Percoll gradient centrifugation revealed these vesicle populations have densities of 1.07 and 1.13 g/ml. The 1.13 g/ml vesicle populations arose during the aggregation stage of differentiation. Although both vesicle populations contained an array of acid hydrolases, they could be shown to differ by several criteria. Electron micrographs of prespore cells showed they contained two types of vesicles with distinct acid phosphatase-staining patterns. One of these vesicle types appeared identical to the lysosomes found in vegetative cells. The second vesicle type had a morphology similar to that of a previously identified organelle, the prespore vesicle. The prespore vesicle is known to contain spore coat proteins which are exocytosed during the final stages of spore differentiation. The higher density acid hydrolase-containing vesicle population was found to contain spore coat proteins. Electron micrographs of the higher density vesicle population showed the presence of acid phosphatase-staining vesicles with a morphology similar to that of prespore vesicles. These data suggest that the higher density acid hydrolase-containing vesicles represent a subpopulation of lysosomes which appear during development and which may be identical to prespore vesicles.

Deoxyglucose-resistant mutants of Neurospora crassa: isolation, mapping, and biochemical characterization.

Neurospora crassa mutants resistant to 2-deoxyglucose have been isolated, and their mutations have been mapped to four genetic loci. The mutants have the following characteristics: (i) they are resistant to sorbose as well as to 2-deoxyglucose; (ii) they are partially or completely constitutive for glucose transport system II, glucamylase, and invertase, which are usually repressed during growth on glucose; and (iii) they synthesize an invertase with abnormal thermostability and immunological properties, suggesting altered posttranslational modification. All of these characteristics could arise from defects in the regulation of carbon metabolism. In addition, mutants with mutations at three of the loci lack glucose transport system I, which is normally synthesized constitutively by wild-type N. crassa. Although the basis for this change is not yet clear, the mutants provide a way of studying the high-affinity system II uncomplicated by the presence of the low-affinity system I.

Isolation and characterization of a Neurospora glucose-repressible gene.

Using differential hybridization, the cDNA copy of a Neurospora gene coding for an abundant glucose-repressible mRNA (grg-1) has been isolated. The cDNA was used to clone the genomic copy, and both were sequenced. The cDNA is nearly full length and contains putative translational start and termination codons. Conceptual translation indicates that grg-1 mRNA could direct the synthesis of a 7,000 molecular weight polypeptide. The genomic clone, contained in an 1,888 bp PvuII fragment, encompasses the entire cDNA as well as 838 bp of 5' and 369 bp of 3' flanking sequence. Comparison of the cDNA and genomic clones revealed the presence of two short introns in potential protein-coding sequences. grg-1 message levels were found to increase within minutes following the onset of glucose deprivation and rise 50 fold during the first 90 min of derepression.

A developmentally controlled change in the post-translational modifications on the lysosomal alpha-mannosidase of the cellular slime mould Dictyostelium discoideum.

During the development of the cellular slime mould Dictyostelium discoideum, a second form of a number of lysosomal enzymes begins to accumulate. The second ('late') form of these enzymes differs from the pre-existing ('early') form in post-translational modification. Pulse-chase experiments using [35S]methionine show that the late form of alpha-mannosidase-1 is made by synthesis de novo starting 8 h after the onset of development. These experiments show there is no interconversion between early and late forms in vivo. A one-dimensional peptide map indicated that the early and late forms of alpha-mannosidase have similar amino acid sequences. The two forms have a similar half-life in vivo when measured during the same period of development. Double-labelling studies were performed with 35SO4 and [3H]leucine or 32PO4 and [3H]leucine. and these studies indicated that the oligosaccharides present on the early form of alpha-mannosidase contained more sulphate and phosphate than did those on the late form. The early enzyme had a 10-fold higher 35S/3H ratio and a 4-fold higher 32P/3H ratio. Endocytosis experiments using early and late alpha-mannoside showed that the early form was efficiently taken up by human fibroblasts, whereas the late form was poorly endocytosed. This suggests that the late form lacks the mannose 6-phosphate residue required for efficient uptake.

Neurospora trehalase and its structural gene.

We have isolated Neurospora trehalaseless mutants and mapped the trehalase structural gene to linkage group I. The structural gene mutations not only affect thermostability and other characteristics of the enzyme but also affect the production of an inhibitor of the wild-type trehalase. The inhibitor appears to be the mutant trehalase. We suggest that the mutant subunits act as inhibitors by entering into the multimeric forms of the enzyme and altering the ability of the normal wild-type subunits to catalyze the cleavage of trehalose.--Wild type trehalase has been purified to near homogeneity, and its characteristics have been studied. It was purified as a tetramer, with each subunit having a molecular weight of 88,000.--We have studied the regulation of trehalase and found the production of trehalase to be glucose repressible. Cells begin to produce trehalase 60 min after being transferred to glucose-free medium.

Neurospora glucamylase and a mutant affected in its regulation.

Neurospora glucamylase is a glucose-repressible extracellular enzyme. The enzyme was purified to homogeneity and found to have a molecular weight of 82,000 and to release glucose from either maltose or amylose. The rate of glucamylase synthesis increases more than 100-fold when cells are transferred from a glucose-containing medium to a glucose-free medium. Increased from a glucose-containing medium to a glucose-free medium. Increased production of glucamylase begins within 30 min of the transfer. Glucamylase is rapidly secreted into the medium. A mutant affecting the ability of glucose to repress the synthesis of the glucose-repressible extracellular enzymes glucamylase and invertase has been isolated and studied. The mutant constitutively synthesizes and secretes a glucamylase which is indistinguishable from the wild-type enzyme.

Changes in the post-translational modification of lysosomal enzymes during development of Dictyostelium.

The form of post-translational modification present on two lysosomal enzymes--acid phosphatase and alpha-mannosidase--changes as part of the developmental program of Dictyostelium discoideum. Prior to 8 h of development, all enzyme molecules are of a single modification type (early form enzyme). Starting at 8 h of development, enzyme molecules with a second type of modification (late-form enzymes) begin to appear in the cell. We separated the early and late forms of these enzymes from each other by chromatography on DEAE-cellulose. We found that the change in protein modification affects the enzymes' in vitro properties. The early and late forms of both of these enzymes differ in thermostability and susceptibility to proteolytic inactivation. We also found that the late form of alpha-mannosidase is preferentially secreted. We suggest that by synthesizing molecules with a second form of modification, the cell confers new characteristics to its lysosomal enzymes.

Protein modification and its biological role.

The modifications present on a polypeptide play an important role in determining its eventual fate. Modifications, particularly proteolysis, are important in the generation of biological activity. Modifications are used to "target" particular polypeptides to specific cellular locations. Protein modification also plays a role in determining the rate of polypeptide degradation. Cells have developed elaborate systems for the modification of their proteins because these modifications serve important biological functions.

Isolation and characterization of Neurospora mutants affected in invertase synthesis.

We have outlined a procedure that allows the large-scale screening of mutagenized Neurospora crassa populations for invertaseless mutants. We have isolated and characterized three mutations, inv(DBL1), inv(DBL9) and inv(DBL14), which have been mapped at or near the invertase structural gene. One of these, inv(DBL1), is particularly interesting. Our experiments indicate that the reduced level of invertase activity in the inv(DBL1)-containing cell can be explained as the result of a reduced number of normal enzyme molecules. We also show that wild-type Neurospora is able to respond rapidly to a change of medium and can dramatically increase its production of invertase within 20 min after a transfer to a carbon-free medium.

Organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of Neurospora.

The organization of the ribosomal DNA (rDNA) repeat unit in the standard wild-type strain of Neurospora crassa, 74-OR23-1A, and in 30 other wild-type strains and wild-collected strains of N. crassa, . tetrasperma, N. sitophila, N. intermedia, and N. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic DNA, and probing of the Southern-blotted DNA fragments with specific cloned pieces of the rDNA unit from 74-OR23-1A. The size of the rDNA unit in 74-OR23-1A was shown to be 9.20 kilobase pairs (kb) from blotting data, and the average for all strains was 9.11 + 0.21 kb; standard error = 0.038; coefficient of variation (C.V.) = 2.34%. These data indicate that the rDNA repeat unit size has been highly conserved among the Neurospora strains investigated. However, while all strains have a conserved HindIII site near the 5' end of the 25 S rDNA coding sequence, a polymorphism in the number and/or position of HindIII sites in the nontranscribed spacer region was found between strains. The 74-OR23-1A strain has two HindIII sites in the spacer, while others have from 0 to at least 3. This restriction site polymorphism is strain-specific and not species-specific. It was confirmed for some strains by restriction analysis of clones containing most of the rDNA repeat unit. The current restriction map of the 74-OR23-1A rDNA repeat unit is presented.

The synthesis of alkaline phosphatase in Neurospora crassa.

Mutations which affect the regulation of Neurospora repressible alkaline phosphatase do so by altering the rate of de novo alkaline phosphatase synthesis. In regulatory mutants the rate of alkaline phosphatase polypeptide synthesis can vary over a 1000-fold range. Following transfer to phosphate-free medium, the wild-type cell is capable of increasing the rate of synthesis of alkaline phosphatase molecules within 30-45 min.