The maize stripe virus major noncapsid protein messenger RNA transcripts contain heterogeneous leader sequences at their 5' termini.

Primer extension analyses and a PCR-based cloning strategy were used to identify and characterize 5' nucleotide sequences on the maize stripe virus (MStV) RNA4 mRNA transcripts encoding the major noncapsid protein (NCP). Direct RNA sequence analysis by primer extension showed that the NCP mRNA transcripts had 10-15 nucleotides beyond the 5' terminus of the MStV RNA4 nucleotide sequence. MStV genomic RNAs isolated from ribonucleoprotein particles (RNPs) lacked the additional 5' nucleotides. cDNA clones representing the 5' region of the mRNA transcripts were constructed, and the nucleotide sequences of the 5' regions were determined for 16 clones. Each was found to have a distinct 10-15 nucleotide sequence immediately 5' of the MStV RNA4 sequence. Eleven of 16 clones had the correct MStV RNA4 5' nucleotide sequence, while five showed minor variations at or near the 5' most MStV RNA4 nucleotide. These characteristics show strong similarities to other viral mRNA transcripts which are synthesized by cap snatching.

Maize stripe virus RNA5 is of negative polarity and encodes a highly basic protein.

The entire 1317 nucleotide sequence of the maize stripe virus (MStV) RNA5 was determined. Only one open reading frame (ORF) was identified and was found in the viral complementary RNA (vcRNA). This ORF appears to encode a protein of M(r) 44237, hereafter referred to as NS5 delta. In vitro translation of transcripts representing nearly full-length RNA5 vcRNA yielded products of the predicted size, as well as some smaller, less prominent products. No products were identified from transcripts of viral polarity. RNA hybridization analyses of MStV-infected Zea mays revealed RNAs corresponding only to full-length RNA5, but both positive and negative polarity RNAs were abundant. Analysis of the NS5 amino acid sequence revealed that it is extremely basic, containing 21% arginine and lysine. Database comparisons showed that NS5 had no significant similarity to other protein sequences.

Complete sequence of maize stripe virus RNA4 and mapping of its subgenomic RNAs.

The complete nucleotide sequence of maize stripe virus RNA4 was determined and found to consist of 2227 nucleotides containing two significant open reading frames. One, in the 5' end of the viral RNA, encodes the major non-capsid protein of M(r) 19,815. The other is located in the 5' end of the viral complementary RNA and could encode a protein of M(r) 31,900. This protein has not been identified previously and has been designated NS4, a non-structural protein. RNA-RNA hybridization detected subgenomic RNAs encoding these proteins, a characteristic of RNA possessing an ambisense gene organization.

Nucleotide sequence and RNA hybridization analyses reveal an ambisense coding strategy for maize stripe virus RNA3.

The 2357-nt sequence of maize stripe virus (MStV) RNA3 was determined. Two nonoverlapping open reading frames (ORFs) of opposite polarities are contained in RNA3. A 591-nt ORF is located near the 5' end of MStV RNA3, while a second ORF of 948 nt is located near the 3' end in the viral complementary RNA (vcRNA). In vitro translation of transcripts derived from cDNA clones representing regions of each ORF was used to identify the respective proteins. A ca. 22,000 Mr protein was translated from the 591-nt ORF transcript. This protein comigrated in SDS-PAGE with a protein produced by in vitro translation of MStV RNA and is referred to as the NS3 protein. The protein from the 948-nt ORF transcript was specifically immunoprecipitated by antiserum to the MStV nucleocapsid protein (N protein). RNA hybridization analyses identified two subgenomic RNAs in total RNA extracts from MStV-infected Zea mays plants. These RNAs are ca. 650 and 1350 nt in size, are of opposite polarities, and correspond to regions of RNA3 containing the two ORFs. These data suggest that MStV RNA3 has an ambisense coding strategy similar to that found for the S RNA of the vertebrate-infecting phleboviruses, uukuviruses, and arenaviruses, as for tomato spotted wilt virus.

Primer extension studies on alpha-amylase mRNAs in barley aleurone. I. Characterization and quantification of the transcripts.

Primer extension was used to characterize alpha-amylase mRNAs from aleurone tissue of barley (Hordeum vulgare L. cv. Himalaya) grains. Two synthetic oligonucleotides, specific for the low-pI and high-pI alpha-amylase groups, were used as primers for synthesis of cDNA from total RNA preparations. Between them, these two oligonucleotides appear to account for all major alpha-amylase mRNAs as judged by hybrid-arrested translation of alpha-amylase mRNAs in a cell-free system. Reconstruction experiments indicated that the levels of extended primers (determined by scintillation counting) were directly proportional to the level of input mRNA over a wide range. This indicates that the technique is suitable for quantification of relative levels of individual alpha-amylase from approximately 2% to 100% of maximal levels. The nucleotide sequences of extended primers defined two different alpha-amylase mRNAs in each of the low-pI and high-pI groups, and possibly a third mRNA in the high-pI group.

Identification and sequence analysis of the maize stripe virus major noncapsid protein gene.

The maize stripe virus (MStV) major noncapsid protein (NCP) gene was characterized, and the location of the NCP gene was identified among the 5-RNA, 18-kb genome. A 12-amino-acid sequence of the NCP was compared with nucleotide sequence data for MStV RNAs 3 and 4 and was found to align perfectly within a 528-nucleotide open reading frame (ORF) of RNA 4. The amino acid composition of purified NCP was almost identical to that deduced from the putative coding region. The deduced NCP molecular weight was 19,815, very similar to that determined by SDS-PAGE analysis of the purified NCP. In vitro transcription and translation analysis of the cDNA representing this region showed unequivocally that this region encoded the NCP. Primer extension analysis using a synthetic oligonucleotide complementary to a sequence near the 5' end of the coding region revealed that the NCP ORF is located 61 nucleotides from the 5' end of RNA 4.

DNA sequence, organization and regulation of the qa gene cluster of Neurospora crassa.

In Neurospora, five structural and two regulatory genes mediate the initial events in quinate/shikimate metabolism as a carbon source. These genes are clustered in an 18 x 10(3) base-pair region as a contiguous array. The qa genes are induced by quinic acid and are coordinately controlled at the transcriptional level by the positive and negative regulators, qa-1F and qa-1S, respectively. The DNA sequence of the entire qa gene cluster has been determined and transcripts for each gene have been mapped. The qa genes are transcribed in divergent pairs and two types of transcripts are associated with each gene: basal level transcripts that initiate mainly from upstream regions and are independent of qa regulatory gene control, and inducible transcripts that initiate downstream from basal transcripts and are dependent on qa-1F binding to a 16 base-pair sequence. We discuss how both types of transcription relate to the organization of the qa genes as a cluster and how this may impose constraints on gene dispersal.

The qa repressor gene of Neurospora crassa: wild-type and mutant nucleotide sequences.

The qa-1S gene, one of two regulatory genes in the qa gene cluster of Neurospora crassa, encodes the qa repressor. The qa-1S gene together with the qa-1F gene, which encodes the qa activator protein, control the expression of all seven qa genes, including those encoding the inducible enzymes responsible for the utilization of quinic acid as a carbon source. The nucleotide sequence of the qa-1S gene and its flanking regions has been determined. The deduced coding sequence for the qa-1S protein encodes 918 amino acids with a calculated molecular weight of 100,650 and is interrupted by a single 66-base-pair intervening sequence. Both constitutive and noninducible mutants occur in the qa-1S gene and two different mutations of each type have been cloned and sequenced. All four mutations occur within the predicted coding region of the qa-1S gene. This result strongly supports the hypothesis that the qa-1S gene encodes a repressor. All four mutations are located within codons for the last 300 amino acids of the qa-1S protein. The mutations in three of the mutants involve amino acid substitutions, while the fourth mutant, which has a constitutive phenotype, contains a frameshift mutation. The two constitutive mutations occur in the most distal region of the gene, possibly implicating the COOH-terminal region of the qa repressor in binding to its target. The two noninducible mutations occur in a region proximal to the constitutive mutations, possibly implicating this region of the qa repressor in binding the inducer.

A leucine tRNA gene adjacent to the QA gene cluster of Neurospora crassa.

A single tRNALeu gene has been localized and sequenced from Neurospora crassa. It is located only 375 bp from the qa gene cluster and it is the only tRNA or 5S rRNA gene within this cloned 37 kb region. The gene encodes a tRNALeu with the anti-codon AAG, and unlike the other nuclear eukaryotic tRNALeu (AAG) gene sequenced (from C. elegans), contains an intervening sequence of 27 bp. The Neurospora tRNALeu (AAG) is 84% and 73% homologous respectively to the C. elegans and bovine tRNALeu (AAG), and is 84% homologous to a Drosophila tRNALeu (CAA). However, it is only 65% homologous to a yeast tRNALeu (CAA) and there is little conservation of intervening sequences or V-loop regions. The gene hybridizes to at least 16 other DNA fragments in the Neurospora genome. Its expression does not seem to be linked to that of the qa genes.

Molecular analysis of the Neurospora qa-1 regulatory region indicates that two interacting genes control qa gene expression.

The qa-1 regulatory region controls the expression of the three structural genes required for the early reactions in quinic acid catabolism in Neurospora crassa. Genetic analysis previously identified two types of noninducible qa-1 mutants, qa-1S and qa-1F, which mapped in separate non-overlapping regions. These mutations were originally interpreted as defining separate domains of a single regulatory protein. This communication describes the further genetic and physical characterization of the qa-1 regulatory region. Using both Neurospora transformation and DNA . RNA hybridization, it has been shown that the qa-1 region consists of two distinct genes corresponding to the two original mutational types qa-1S and qa-1F. The analysis of the mRNA species hybridizing to these regions indicates that the qa-1F gene encodes a 2.9-kilobase (kb) mRNA, while the qa-1S gene encodes related 4.1-kb and 3.4-kb mRNAs. The transcriptional regulation of one of these genes, qa-1S, was examined. Evidence is presented that the qa-1S gene is induced by quinic acid and is also subject to apparent autogenous regulation as well as to control by the qa-1F gene product. Based on these results and earlier genetic analysis, the hypothesis is proposed that one of the two qa regulatory genes encodes a repressor protein (qa-1S), and the other encodes an activator protein (qa-1F), both of which control qa gene expression.