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J P Mascarenhas

Publications and source records attributed to J P Mascarenhas.

At least 19 recordsLinked to original sources
PubMed1998

A monocot pollen-specific promoter contains separable pollen-specific and quantitative elements.

The region of the promoter of the pollen-specific maize gene, ZM13, from -119 to -37 was analyzed by a linker-scanning type of substitution mutagenesis and two areas were shown to be responsible for pollen expression: a proximal region delineated by mutations from -84 to -53 that conferred pollen specificity, and an upstream region delineated by a mutation from -107 to -102 (Q-element) that could increase the expression of the proximal region but showed no ability to cause expression in pollen on its own. Replacement of both of these areas with other sequences including the CaMV 35S promoter failed to replace activity.

Allergens↗
PubMed1996

Expression of Zm13, a pollen specific maize protein, in Escherichia coli reveals IgE-binding capacity and allergenic potential.

Plant proteins belong to the most frequent elicitors of type I allergic symptoms in industrialized countries. Several relevant plant allergens have been found to be either specifically expressed or highly upregulated in mature pollen. The cDNA coding for a pollen specific maize protein, Zm13, shows significant sequence homology with a number of pollen or anther specific proteins from monocot and dicot plants as well as with recently described allergens from olive and rye grass. To test whether the Zm13 protein might possess IgE-binding capacity, Zm13 was expressed in E. coli. The coding region of Zm13 was PCR amplified from a genomic clone and expressed as a glutathione-S-transferase fusion protein. The recombinant Zm13 fusion protein bound a Zm13 specific rabbit antiserum and reacted with serum IgE from grass pollen allergic patients indicating that Zm13 and homologous proteins represent a family of conserved plant allergens.

Allergens↗
PubMed1993

Isolation and characterization of pollen-specific maize genes with sequence homology to ragweed allergens and pectate lyases.

A cDNA clone (Zm58.1) was isolated by differential screening from a cDNA library made to mature Zea mays pollen, and shown to be pollen-specific by RNA blot analysis. When this partial-length clone was used to probe a genomic library, a similar but distinct pollen-specific genomic clone (68% sequence identity) was isolated (Zm58.2). The putative proteins coded for by these two clones show sequence homology to several flower-expressed gene products from various plant species, including known pollen allergens from short ragweed (Ambrosia artemisiifolia), and to pectate lyases from the plant pathogenic bacteria Erwinia spp. The two genes map to different chromosomes.

Allergens↗
PubMed1992

Dissection of a pollen-specific promoter from maize by transient transformation assays.

We have previously reported the isolation and characterization of a gene (Zm13) from Zea mays which shows a pollen-specific pattern of expression. Stably transformed tobacco plants containing a reporter gene linked to portions of the Zm13 5' flanking region show correct temporal and spatial expression of the gene. Here we present a more detailed analysis of the 5' regions responsible for expression in pollen by utilizing a transient expression system. Constructs containing the beta-glucuronidase (GUS) gene under the control of various sized fragments of the Zm 13 5' flanking region were introduced into Tradescantia and Zea mays pollen via high-velocity microprojectile bombardment, and monitored both visually and with a fluorescence assay. The results suggest that sequences necessary for expression in pollen are present in a region from -100 to -54, while other sequences which amplify that expression reside between -260 and -100. The replacement of the normal terminator with a portion of the Zm13 3' region containing the putative polyadenylation signal and site also increased GUS expression. While the -260 to -100 region contains sequences similar to other protein-binding domains reported for plants, the -100 to -54 region appears to contain no significant homology to other known promoter fragments which direct pollen-specific expression. The microprojectile bombardment of Tradescantia pollen appears to be a good test system for assaying maize and possibly other monocot promoter constructs for pollen expression.

Gene Expression↗
PubMed1990

Promoter sequences from a maize pollen-specific gene direct tissue-specific transcription in tobacco.

A set of 5' promoter deletions from Zmg13, a genomic clone of a pollen-specific gene of maize, has been transcriptionally fused to a beta-glucuronidase (GUS) reporter gene in the binary vector pBI101. Tobacco leaf disks were transformed and mature plants analyzed for GUS activity directed by the Zmg13 promoter constructs. Transgenic plants containing the 375 bp Zmg13 sequence from -314 to +61 relative to the transcription start site transcribed GUS RNA and expressed active GUS enzyme in mature pollen but not in leaves. Plants transformed with a 35S CaMV promoter-GUS transcriptional fusion expressed GUS RNA in leaves but not in pollen. Neither GUS RNA or active enzyme could be detected in pollen or leaves from plants containing a 124 bp Zmg13-GUS transcriptional fusion missing the putative Zmg13 TATA box. No GUS RNA or enzyme expression was not detected in non-transformed tobacco. RNA and GUS histochemical analysis of the T1 generation confirmed that the temporal expression pattern of Zmg13-GUS transcription in tobacco followed that of the native gene in maize and that the Zmg13 promoter sequences from the maize gene are able correctly to direct genetically stable, tissue-specific gene expression in transgenic tobacco plants.

Base Sequence↗
PubMed1989

Characterization of a pollen-specific cDNA clone from Zea mays and its expression.

A pollen-specific cDNA clone, Zmc13, has been isolated from a cDNA library constructed to poly(A) RNA from mature maize pollen. The cDNA as shown by primer extension analysis is a full-length copy of the mRNA. The cDNA has been sequenced and is 929 nucleotides in length plus a 47-nucleotide poly(A) tail. Putative polyadenylation signals are identifiable in the 3'-nontranslated region. The mRNA codes for a predicted polypeptide containing 170 amino acid residues and with a molecular mass of 18.3 kilodaltons. The hydropathy profile suggests a possible signal sequence on the amino terminus. A comparison of the nucleotide and deduced amino acid sequence with sequences in data banks has not shown homology to known molecules. In situ hybridizations using RNA probes show that the mRNA is located in the cytoplasm of the vegetative cell of the pollen grain and after germination is distributed throughout the pollen tube cytoplasm.

Amino Acid Sequence↗
PubMed1987

Genes expressed in the male gametophyte of flowering plants and their isolation.

Recombinant cDNA libraries to poly(A)RNA isolated from mature pollen of Zea mays and Tradescantia paludosa have been constructed. Northern blot analyses indicate that several of the clones are unique to pollen and are not expressed in vegetative tissues. The majority, however, are expressed both in pollen and vegetative tissues. Southern hybridizations show that the pollen specific sequences in corn are present in one or a very few copies in the genome. By using several of the clones as probes, it was found that there are at least two different groups of mRNAs with respect to their synthesis. The mRNAs of the first group represented by the pollen specific clones are synthesized after microspore mitosis and increase in concentration up to maturity. The second group, exemplified by actin mRNA, begins to accumulate soon after meiosis, reaches its maximum by late pollen interphase, and decreases thereafter. Although the actin mRNA and the pollen specific mRNAs studied show very different patterns of initiation of synthesis and accumulation during pollen development, the rates of decline of these mRNAs during the first 60 minutes of germination and pollen tube growth in Tradescantia are similar and reflect the previously observed declines in rates of protein synthesis during this period.

Journal Article↗
PubMed1985

Onset of Alcohol Dehydrogenase Synthesis during Microsporogenesis in Maize.

During male gametophyte development the synthesis of several proteins occurs from transcripts of the haploid genome. Alcohol dehydrogenase (ADH1), a developmentally regulated protein, was chosen for study to determine the stage at which its synthesis was initiated and the pattern of its synthesis during microsporogenesis. The ability of ADH to reduce p-nitro blue tetrazolium chloride in situ was used as an indicator of enzyme activity. Maize strains heterozygous for adh1 were utilized to provide an internal control, 50% of the grains being adh1(+) and 50% being adh1(-). No ADH activity was detectable when tetrads were first formed after meiosis. Activity was initially detected soon after the tetrads began to break apart but before the microspores in the tetrads had completely separated. The transcription of the adh1 gene from the haploid genome must thus occur very soon after meiosis is completed. ADH activity increases at a constant rate thereafter until microspore mitosis when an increase in the rate takes place which lasts until generative cell division. Thereafter, there is a marked decrease in the rate of accumulation of ADH activity.

Journal Article↗
PubMed1985

High temperature-induced thermotolerance in pollen tubes of tradescantia and heat-shock proteins.

Growing pollen tubes of Tradescantia paludosa are protected from inhibition of growth at 41 degrees C by a prior exposure to gradually increasing temperatures. Heat shock proteins (hsps) are not synthesized by pollen tubes as determined by labeling with [(35)S]methionine and two-dimensional gel electrophoresis, during either a heat shock at 41 degrees C or a gradual temperature increase to 41 degrees C. A comparison after two-dimensional electrophoresis of silver-stained spots and radioactive spots after autoradiography of an extract of ungerminated pollen mixed with a trace amount of [(35)S]methionine-labeled vegetative tissue heat shocked at 41 degrees C to act as a hsps marker, indicates that the majority, if not all, of the major hsps are not present in the pollen grain at anthesis. The type of thermotolerance seen with pollen tubes can thus be achieved without the presence or the new synthesis of the hsps.

Journal Article↗
PubMed1984

Analysis of the Complexity and Diversity of mRNAs from Pollen and Shoots of Tradescantia.

The mRNAs of the mature pollen grain of Tradescantia paludosa at anesthesia and of vegetative shoots have been compared by analyzing the kinetics of hybridization between homologous and heterologous reactions of cDNA to poly(A)RNA in excess. The mRNAs in pollen can be divided into three abundance classes with complexities of 5.2 x 10(4), 1.6 x 10(6), and 2.1 x 10(7) nucleotides. The three classes are made up of sequences that constitute 15, 60, and 24% of the mRNAs and each sequence is present on an average at 26,000, 3,400, and 100 copies, respectively, per pollen grain. About 20,000 different genes are expressed in pollen as compared to about 30,000 in vegetative shoots. Estimates have been made of pollen mRNA sequences shared with those of shoot tissue and of shoot sequences common to those in pollen.

Journal Article↗
PubMed1981

Microspore and microgametophyte development in relation to biological activity of environmental pollutants.

The pattern of synthesis of nucleic acids and proteins in the development of the microspore and later during pollen germination and tube growth is discussed. In the pollen grain at the time of anthesis all the proteins that are required for germination and early tube growth are either already present, or if new proteins are synthesized, the messenger RNAs for their synthesis already exist in the ungerminated pollen grain. In addition, similar proteins are synthesized on new mRNAs during germination and pollen tube growth as are synthesized on premade mRNAs. The genetic program during at least the latter part of pollen maturation prior to anthesis is thus the same as that during pollen germination and tube growth. Accordingly, one cannot treat mature pollen with mutagens and expect to be able to score the pollen tubes for mutant proteins. Treatment with mutagenic compounds would have to be during pollen maturation in the anther, before the transcription for the proteins required during germination and pollen tube growth has occurred. Available evidence indicates that this is very early in pollen development, possibly soon after meiosis. The value of pollen tubes to monitor for chemicals that affect the intracellular motility systems of organisms is also discussed.

Cytoplasmic Streaming↗
PubMed1978

RNA synthesis in whole cells and protoplasts of centaurea: a comparison.

Protoplasts enzymically isolated from suspension cultures of Centaurea cyanus L. incorporate radioactive precursors into RNA with kinetics similar to that of whole cells. There are differences, however, in several other aspects of RNA metabolism. The proportion of total RNA that contains poly(A) sequences (25 to 30%) is similar in both freshly isolated protoplasts and whole cells after a 20-minute pulse with [(3)H]adenosine. After a 4-hour pulse, however, poly(A)-containing RNA makes up 30% of the total RNA in protoplasts whereas it drops to 8% in whole cells. There appears to be a faulty processing of ribosomal precursor into the mature ribosomal species, as the precursor seems to accumulate to higher levels relative to the mature 18S and 25S rRNAs in protoplasts as compared to whole cells. Additional differences are seen in the size distributions of poly(A)-containing RNA, although the length of the poly(A) segment is similar in both protoplasts and whole cells. Within 24 hours protoplasts appear to have resumed a pattern of RNA synthesis similar to that of whole cells.

Journal Article↗