Pearl millet cysteine protease inhibitor. Evidence for the presence of two distinct sites responsible for anti-fungal and anti-feedent activities.
Recently, pearl millet cysteine protease inhibitor (CPI) was, for the first time, shown to possess anti-fungal activity in addition to its anti-feedent (protease inhibitory) activity [Joshi, B.N. et al. (1998) Biochem. Biophys. Res. Commun. 246, 382-387]. Characterization of CPI revealed that it has a reversible mode of action for protease inhibition. The CD spectrum exhibited a 35% alpha helix and 65% random coil structure. The intrinsic fluorescence spectrum was typical of a protein devoid of tryptophan residues. Demetallation of Zn2+ resulted in a substantial change in the secondary and tertiary structure of CPI accompanied by the complete loss of anti-fungal and inhibitory activity indicating that Zn2+ plays an important role in maintaining both structural integrity and biological function. The differential response of anti-fungal and inhibitory activities to specific modifiers showed that there are two different reactive sites associated with anti-fungal and anti-feedent activity in CPI located on a single protein as revealed from its N-terminal sequence data (AGVCYGVLGNNLP). Modification of cysteine, glutamic/aspartic acid or argnine resulted in abolition of the anti-fungal activity of CPI, whereas modification of arginine led to an enhancement of the inhibitory activity in solution. Modification of histidine resulted in a twofold increase in the protease inhibitory activity without affecting the anti-fungal activity, whereas modification of serine led to selective inhibition of the protease inhibitory activity. The differential nature of the two activities was further supported by differences in the temperature stabilities of the anti-fungal (60 degrees C) and inhibitory (40 degrees C) activities. Binding of papain to CPI did not abolish the anti-fungal activity of CPI, supporting the presence of two active sites on CPI. The differential behavior of CPI towards anti-fungal and anti-feedent activity cannot be attributed to changes in conformation, as assessed by their CD and fluorescence spectra. The interaction of CPI modified for arginine or histidine with papain resulted in an enhancement of CPI activity accompanied by a slight decrease in fluorescence intensity of 15-20% at 343 nm. In contrast, modification of serine resulted in inhibition of CPI activity with a concomitant increase of 20% in the fluorescence intensity when complexed by the enzyme. This implies the involvement of enzyme-based tryptophan in the formation of a biologically active enzyme-inhibitor complex. The presence of anti-fungal and anti-feedent activity on a single protein, as evidenced in pearl millet CPI, opens up a new possibility of raising a transgenic plant resistant to pathogens, as well as pests, by transfer of a single CPI gene. (+info)
A gene cluster encoded by panicum mosaic virus is associated with virus movement.
A subgenomic RNA (sgRNA) of about 1500 nucleotides has been detected in millet plants and protoplasts infected with panicum mosaic virus (PMV). This sgRNA expressed p8, p6.6, p15, and the 26-kDa capsid protein (CP) genes during in vitro translation assays, as determined by using mutants inactivated for expression of each open reading frame. Abolishing expression of p8 and p6.6, the two 5'-proximal genes on the sgRNA, did not affect the replication of PMV in millet protoplasts, but obstructed spread in plants. As predicted for a typical cell-to-cell movement protein, p8 localized to the cell wall fraction of PMV-infected millet plants. The introduction of premature stop codons downstream of the PMV p15 start codon (p15*) abolished infectivity in planta, but did not impair replication in protoplasts. However, a delayed systemic infection in millet plants was supported by the p15aug(-) start codon mutant, which may reflect very low levels of expression from a suboptimal start codon context and/or leaky scanning to a second inframe AUG codon to express the C-terminal portion of the 15-kDa protein. PMV CP mutants had little effect on sgRNA accumulation, but were correlated with a reduction of the gRNA and the decreased expression of the 8-kDa protein in protoplasts as well as abolishment of cell-to-cell spread in plants. These results imply that the successful establishment of a PMV systemic infection in millet host plants appears to be dependent on the concerted expression of the p8, p6.6, p15, and CP genes. (+info)
Endemic goiter with iodine sufficiency: a possible role for the consumption of pearl millet in the etiology of endemic goiter.
BACKGROUND: Deficiencies of iodine, iron, and vitamin A are the 3 most common micronutrient deficiencies in developing countries, although control programs, when properly implemented, can be effective. OBJECTIVE: We investigated these deficiencies and their possible interaction in preschool children in the southern Blue Nile area of Sudan. DESIGN: Goiter, signs of vitamin A deficiency, and biochemical markers of thyroid, vitamin A, and iron status were assessed in 984 children aged 1-6 y. RESULTS: The goiter rate was 22. 3%. The median urinary iodine concentration was 0.79 micromol/L and 19.3% of the children had a concentration >1.57 micromol/L. Although serum thyroxine and triiodothyronine concentrations were within reference ranges, the median thyrotropin concentration was 3.78 mIU/L and 44% of the children had thyrotropin concentrations above normal. The mean urinary thiocyanate concentration was high (259 +/- 121 micromol/L). The prevalences of Bitot spots and night blindness were 2.94% and 2.64%, respectively, and 32% of the subjects had serum retinol binding protein concentrations <15 mg/L. A significant positive correlation was observed between thyrotropin and retinol binding protein. Whereas 88% of the children had hemoglobin concentrations <1.86 mmol/L, only 13.5% had serum ferritin concentrations below the cutoff of 12 microg/L and 95% had serum transferrin concentrations above the cutoff of 2.50 g/L. CONCLUSIONS: Our results indicate that goiter is endemic in this region of Sudan despite iodine sufficiency and that both anemia and vitamin A deficiency are health problems in the area. Moreover, consumption of millet, vitamin A deficiency, and protein-energy malnutrition are possible etiologic factors in this endemic area. (+info)
Polypeptide compositions and NH2-terminal amino acid sequences of proteins in foxtail and proso millets.
Seed protein of foxtail and proso millets were fractionated into polypeptides that were analyzed for their major protein, prolamin, and the NH2-terminal amino acid sequences of the proteins were determined. The proteins extracted from foxtail and proso millets were 64.1% and 80.0% prolamin, respectively. The polypeptides of the prolamins were classified into two groups. The major polypeptides of 27-19 kDa were rich in leucine and alanine, whereas the 17-14 kDa polypeptides were rich in methionine and cysteine. Glutelin-like proteins that were extracted with a reducing reagent were high in proline content, the major polypeptides being 17 and 20 kDa. The NH2-terminal amino acid sequence showed that the major polypeptides of prolamin were homologous to alpha-zein and a glutelin-like protein containing the Pro-Pro-Pro sequence, like the repetitive sequence of gamma-zein. Although the prolamin consisted of a similar subunit to that of zein, polypeptides with various pI values were found among them. (+info)
In vitro- and in vivo-generated defective RNAs of satellite panicum mosaic virus define cis-acting RNA elements required for replication and movement.
Satellite panicum mosaic virus (SPMV) depends on its helper virus, panicum mosaic virus (PMV), to provide trans-acting proteins for replication and movement. The 824-nucleotide (nt) genome of SPMV possesses an open reading frame encoding a 17.5-kDa capsid protein (CP), which is shown to be dispensable for SPMV replication. To localize cis-acting RNA elements required for replication and movement, a comprehensive set of SPMV cDNA deletion mutants was generated. The results showed that the 263-nt 3' untranslated region (UTR) plus 73 nt upstream of the CP stop codon and the first 16 nt in the 5' UTR are required for SPMV RNA amplification and/or systemic spread. A region from nt 17 to 67 within the 5' UTR may have an accessory role in RNA accumulation, and a fragment bracketing nt 68 to 104 appears to be involved in the systemic movement of SPMV RNA in a host-dependent manner. Unexpectedly, defective RNAs (D-RNAs) accumulated de novo in millet plants coinfected with PMV and either of two SPMV mutants: SPMV-91, which is incapable of expressing the 17.5-kDa CP, and SPMV-GUG, which expresses low levels of the 17.5-kDa CP. The D-RNA derived from SPMV-91 was isolated from infected plants and used as a template to generate a cDNA clone. RNA transcripts derived from this 399-nt cDNA replicated and moved in millet plants coinoculated with PMV. The characterization of this D-RNA provided a biological confirmation that the critical RNA domains identified by the reverse genetic strategy are essential for SPMV replication and movement. The results additionally suggest that a potential "trigger" for spontaneous D-RNA accumulation may be associated with the absence or reduced accumulation of the 17.5-kDa SPMV CP. This represents the first report of a D-RNA associated with a satellite virus. (+info)
Geographical variation of the alleles at the two prolamin loci, Pro1 and Pro2, in foxtail millet, Setaria italica (L.) P. Beauv.
Allelic variation at the two prolamin loci (Pro1 and Pro2) and its geographical distribution in 560 local cultivars of foxtail millet (Setaria italica) mainly from Eurasia were studied using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Genetic analysis of a newly detected polymorphic band, band 6, indicated that it is controlled by an allele at the Pro2 locus, which was designated as Pro2f. Two alleles (Pro1a and Pro1null) at the Pro1 locus and six alleles (Pro2a, Pro2b, Pro2c, Pro2d, Pro2e and Pro2f) at the Pro2 locus were detected among the cultivars examined. Although the frequency of the Pro1a allele varied from 0% in the Nansei islands of Japan and Africa to 66% in Afghanistan, no apparent trend was observed in geographical distribution. In contrast, two common alleles at the Pro2 locus, Pro2b and Pro2f, had clear differential geographical distribution. The Pro2b allele was most frequent in Europe and decreased in frequency eastwards. The Pro2f allele occurred frequently in subtropical and tropical regions including the Nansei islands of Japan, the Philippines, Nepal, India, Pakistan and Africa. All eight alleles at the Pro1 and Pro2 loci occurred in China, suggesting China is a center of diversity. The origin of geographical differentiation of local cultivars into a "tropical group" characterized by the Pro2f allele and other genes was discussed. (+info)
N-terminal amino acid sequences of prolamins encoded by the alleles at the Pro1 and Pro2 loci in foxtail millet, Setaria italica (L.) P. Beauv.
N-terminal amino acid sequences of six prolamins encoded by seven alleles at two loci, Pro1 and Pro2, of foxtail millet (Setaria italica (L.) P. Beauv.) were analyzed and compared with other prolamins of subfamily Panicoideae. Based on the N-terminal amino acid sequences, band 3 (the prolamin purified from band 3) which is controlled by an allele at the Pro1 locus and bands 1, 2, 4, 5 and 6 which are controlled by alleles at the Pro2 locus could be classified into three groups. Band 3 was found to be homologous to the prolamin of pearl millet (Pennisetum americanum) and is designated as the "pennisetin-like prolamin". Bands 2 and 4, and bands 1, 5 and 6 were subdivided into "x-type prolamin" and "y-type prolamin". Both of the x-type and y-type prolamins showed homology with prolamin of Echinochloa crus-galli and alpha-zein-like prolamins of maize, sorghum and Job's tears. Therefore, these prolamins were designated as "alpha-zein-like prolamin". These results suggest that alleles at the Pro1 locus and those at the Pro2 locus have not arisen from an identical ancestral gene, and that the Pro2 locus comprise two tightly linked genes, which encode similar prolamins. Hypotheses on the diversification of alleles at the Pro2 locus are discussed based on the N-terminal amino acid sequences of the respective bands, combinations of bands controlled by the alleles, and frequencies of the alleles. (+info)
RNA: protein interactions associated with satellites of panicum mosaic virus.
The interactions between satellite panicum mosaic virus (SPMV) capsid protein (CP) and its 824 nucleotide (nt) single stranded RNA were investigated by gel mobility shift assay and Northwestern blot assay. SPMV CP has specificity for its RNA at high affinity, but little affinity for non-viral RNA. The SPMV CP also bound a 350 nt satellite RNA (satRNA) that, like SPMV, is dependent on panicum mosaic virus for its replication. SPMV CP has the novel property of encapsidating SPMV RNA and satRNA. Competition gel mobility shift assays performed with a non-viral RNA and unlabeled SPMV RNA and satRNA revealed that these RNA:protein interactions were in part sequence specific. (+info)