Biomedical subjects

B A Larkins

Publications and source records attributed to B A Larkins.

At least 19 recordsLinked to original sources

PubMed1992

Evidence for the translational control of storage protein gene expression in oat seeds.

We employed a rapid fractionation method coupled with a sensitive enzyme-linked immunosorbent assay to quantify the globulins and avenins in developing and mature oat seeds. On a molar basis, there is approximately 10-11 times as much globulin as avenin. Pulse labeling of endosperm proteins indicated that the rate of globulin synthesis is approximately nine times that of avenin. In addition, neither protein class showed any signs of degradation during this experiment. Analysis of the storage protein mRNAs indicates that both globulin and avenin transcripts are associated with membrane-bound polysomes and are found in similar concentrations within the membrane-bound polysome fraction. We found that avenin and globulin mRNAs are fully loaded with ribosomes, suggesting that initiation is not rate-limiting for translation of either protein. Rates of globulin and avenin synthesis were similar when synthetic storage protein mRNAs were translated in vitro. Translation of equimolar amounts of globulin and avenin mRNAs in the same reaction showed equivalent amounts of protein synthesized when compared with globulin and avenin mRNAs translated in separate reaction mixes. We propose that translation elongation or termination reactions are likely regulatory steps for controlling storage protein synthesis in oat endosperm.

Autoradiography↗

PubMed1992

Sequence analysis of linked maize 22 kDa alpha-zein genes.

We have determined the nucleotide sequence of a 7343 bp zein genomic clone (gZ22.8H3) from the maize inbred W64A. Computer-aided analysis of the DNA sequence revealed two contiguous 22 kDa alpha-zein genes. The 5' gene (gZ22.8) encodes a complete polypeptide and contains putative regulatory sequences in both the 5' and 3' flanking regions that are typical of zein genes. In contrast, the 3' gene (psi gZ22.8) appears to be a pseudogene, because it contains numerous insertions and deletions that would prevent translation of the mRNA. Alignment of the 5' and 3' flanking sequences of both genes indicated that they resulted from a 3.3 kb DNA duplication event.

Amino Acid Sequence↗

PubMed1992

Nucleotide sequence analysis of the genomes of the MAV-PS1 and P-PAV isolates of barley yellow dwarf virus.

The MAV-PS1 and P-PAV isolates of barley yellow dwarf virus (BYDV) are serologically related, but not identical. Both are transmitted by the aphid Macrosiphum avenae, but P-PAV is also transmitted by Rhopalosiphum padi. To evaluate the basis for these and other differences, overlapping clones from cDNA libraries representing the genome of each isolate were characterized by restriction enzyme digestion and by hybridization, and subsequently sequenced. Each genome has six positive strand open reading frames (ORFs) which are similar to those identified from a BYDV isolate from Australia (Vic-PAV). The greatest diversity between MAV-PS1 and P-PAV sequences was found in ORFs located in the 3' half of the respective genomes, in particular ORFs 5 and 6, suggesting that these regions of the genome may be involved in the properties that differentiate MAV-PS1 and P-PAV. Sequence comparisons between P-PAV and Vic-PAV showed a high degree of identity in that all ORFs showed greater than 90% amino acid similarity, except ORF6 which had only 69% similarity.

Amino Acid Sequence↗

PubMed1992

Pumpkin phloem lectin genes are specifically expressed in companion cells.

Pumpkin phloem exudate contains two abundant phloem proteins: PP1 is a 96-kD protein that forms polymeric filaments in vivo, and PP2 is a 48-kD dimeric lectin. Polyclonal antibodies raised against pumpkin phloem exudate were used to isolate several cDNAs corresponding to PP1 and PP2. RNA gel blot analysis indicated that PP1 is encoded by an mRNA of approximately 2500 nucleotides, whereas PP2 subunits are encoded by an mRNA of 1000 nucleotides. Sequence analysis of PP2 cDNAs revealed a 654-bp open reading frame encoding a 218-amino acid polypeptide; this polypeptide had the carbohydrate binding characteristics of a PP2 subunit. The PP2 mRNA was localized within the phloem of pumpkin hypocotyl cross-sections based on in situ hybridization of a digoxigenin-labeled antisense probe. PP2 mRNA was found within the companion cells in both the bicollateral vascular bundles and the extrafascicular phloem network.

Amino Acid Sequence↗

PubMed1991

Normal and lysine-containing zeins are unstable in transgenic tobacco seeds.

Chimeric genes composed of the beta-phaseolin promoter, an alpha-zein coding sequence and its modified versions containing lysine codons, and a beta-zein polyadenylation signal were inserted into the genome of tobacco by Agrobacterium-mediated transformation. alpha-Zein mRNA levels in the transgenic tobacco seeds 20 days after self-pollination varied between 1.0% and 2.5% of the total mRNA population. At 25 days after pollination the 19 kDa alpha-zein was immunologically detected with a polyclonal antiserum in protein extracts from the seeds of transgenic plants. The transgenic plant with the highest level of zein gene expression had an alpha-zein content that was approximately 0.003% of the total seed protein. The amount of alpha-zein in other transgenic plants varied between 1 x 10(-4)% and 1 x 10(-5)% of the total seed protein. The differences in the amounts of mRNA and protein did not correlate with the lysine substitutions introduced into the alpha-zein protein. Polysomes translating alpha-zein mRNA isolated from tobacco seeds contained fever ribosomes than those from maize endosperm, but this did not appear to be the cause of the inefficient protein synthesis. In vivo labelling and immunoprecipitation indicated that newly synthesized alpha-zein was degraded in tobacco seeds with a half-life of less than 1 hour.

Lysine↗

PubMed1991

Binding of an endosperm-specific nuclear protein to a maize beta-zein gene correlates with zein transcriptional activity.

Promoter regions of alpha- and beta-zein genes were analyzed for binding of nuclear proteins from developing endosperm and seedling tissue of maize. Using a band-shift assay, we identified two distinct protein factors, alpha-1 and beta-1, that interacted specifically with alpha- and beta-zein gene promoter regions, respectively. Alpha-1 was present in nuclei from both endosperm and seedling tissue, whereas beta-1 was found only in nuclei from developing endosperm tissue. Mixing of nuclear extracts demonstrated that seedling tissue contained undetectable amounts of beta-1, rather than having an inhibitor for formation of the beta-1/DNA complex. Chemical footprinting analysis localized the beta-1 recognition site to a 22 bp sequence flanked by CCAT and TATA boxes. The apparent molecular mass of beta-1 was determined to be 29 kDa by southwestern blotting. Based on in vitro binding assays, the greatest concentration of the beta-1 in endosperm nuclei is at 16 days after pollination, which coincides with the time of highest transcriptional activity of the beta-zein gene. These results suggest that beta-1 may act as a tissue-specific, trans-acting regulator of the expression of the beta-zein gene in developing maize endosperm.

Base Sequence↗

PubMed1991

Nucleotide sequence analysis and genomic organization of the NY-RPV isolate of barley yellow dwarf virus.

cDNA clones representing the ssRNA genome of the NY-RPV isolate of barley yellow dwarf luteovirus (BYDV) were sequenced and 5600 nucleotides of the genome were determined. The deduced genome organization has limited similarity to that of another BYDV isolate, Vic-PAV, but is identical to that of beet western yellows (BWYV) and potato leafroll (PLRV) luteoviruses. NY-RPV has six major positive-sense open reading frames (ORFs) and, by comparison with RNA-dependent RNA polymerase and nucleic acid helicase consensus sequence motifs, it is postulated that NY-RPV ORF2 and ORF3 encode the viral replicase, which is expressed by a translational frameshift mechanism. The region of the NY-RPV genome containing the 22K coat protein ORF, the apparently associated internal apparent VPg ORF and the ORF immediately 3'-proximal (ORF6) to the coat protein ORF are organized as reported for other luteoviruses. Evidence is presented showing that ORF6 is expressed by readthrough of the coat protein gene termination codon, and that this protein is associated with the intact virus as a 65K protein. Although NY-RPV infects graminaceous rather than dicotyledonous plants, the taxonomic relationships between BYDV isolates and other luteoviruses deduced from the genome organization and sequence data strongly suggest that NY-RPV is distinct from the PAV-like isolates of BYDV and is more closely related to BWYV and PLRV.

Amino Acid Sequence↗

PubMed1991

opaque-2 modifiers increase gamma-zein synthesis and alter its spatial distribution in maize endosperm.

Through the action of opaque-2 modifier genes, the soft, floury endosperm of opaque-2 mutants is converted to a vitreous phenotype. This change in endosperm texture is associated with a twofold to threefold increase in gamma-zein content. To investigate the effect of opaque-2 modifiers on the expression of gamma-zein genes, we analyzed the synthesis and distribution of gamma-zein protein and the level of gamma-zein mRNAs in developing endosperms of the inbreds W64A and W64Ao2, a modified opaque-2 mutant Pool 34 QPM, and their reciprocal F1 hybrids. We also characterized the number and organization of gamma-zein genes in these and related maize genotypes. Our studies show that opaque-2 modifiers are semidominant genes, resulting in a twofold to threefold increase in gamma-zein gene expression in both opaque-2 and normal genetic backgrounds. The increase in gene expression appears to be a consequence of enhanced mRNA transcription or stability rather than gene amplification because gamma-zein genes occur in one or two copies in modified as well as nonmodified genetic backgrounds. Ultrastructural studies showed that gamma-zein occurs in high concentrations in the first few subaleurone cells of nonmodified endosperms, but high concentrations of gamma-zein occur in the subaleurone and central endosperm cells of modified opaque-2 mutants. The increased concentration and distribution of gamma-zein in modified endosperms are highly correlated with the activity of opaque-2 modifier genes.

Microscopy, Immunoelectron↗

PubMed1990

Analysis of seed storage protein genes of oats.

We have isolated genomic clones encoding the two major classes of seed storage proteins in oats, the 12 S globulins and the avenins. The globulin genes encode glutamine-rich, sulfur-poor storage proteins that are highly conserved in sequence and structure. The globulin genes contain three short introns whose positions in the coding sequence are the same as in storage globulin genes in legumes and other dicots. The avenin genomic clone contains four tightly linked genes that belong to both of the two avenin gene subfamilies. The avenin genes encode glutamine-rich, lysine-poor proteins that vary in length due to differences in the number of peptide repeats. Although globulin and avenin genes are expressed coordinately during oat seed development, their promoter regions do not contain any conserved sequence elements that might determine developmental timing. Previous studies showed that there are roughly equal amounts of globulin and avenin mRNAs in developing oat seed, despite there being much more globulin than avenin in mature seed. Storage protein synthesis in oats must therefore be controlled partially by post-transcriptional mechanisms. Sequence analysis of globulin and avenin genes has provided several clues as to why globulin mRNAs may be translated more efficiently than avenin mRNAs.

Allergens↗

PubMed1990

Analysis of promoter activity from an alpha-zein gene 5' flanking sequence in transient expression assays.

Three DNA regions required for high levels of transcription were identified by transient gene expression analysis of the 5' flanking region of a 19 kDa alpha-zein gene. For these analyses, the zein promoter region was fused to the beta-glucuronidase (GUS) gene and assayed by transient expression in carrot protoplasts. A 107-bp sequence (-114/-8) containing the TATA box resulted in low levels of GUS activity. Addition of the proximal 75 bp (-189/-114) doubled the level of GUS expression, and a further increase in expression was obtained when additional upstream sequences (-483/-226) were placed 5' of the zein promoters. Zein upstream sequences enhanced transcription independently of the -189/-114 region. Although the -189/-114 region was not essential for transcription, it was important to obtain maximum GUS activity. A 121 bp upstream sequence (-347/-226) that contains the conserved TGTAAAG sequence gave high levels of GUS activity when placed in either orientation 5' of the zein promoter sequences. However, nucleotides -347 to -309, containing the TGTAAAG sequence, could be deleted from this fragment without a significant change in GUS activity. Zein upstream sequences did not promote transcription of the GUS gene in somatic maize protoplasts. The upstream activating sequence from the cauliflower mosaic virus (CaMV) 35S promoter placed 5' of deletion mutants of the zein promoter also failed to produce GUS activity above background.

Base Sequence↗

PubMed1990

Nucleotide sequences of coat protein genes for three isolates of barley yellow dwarf virus and their relationships to other luteovirus coat protein sequences.

Barley yellow dwarf virus (BYDV) can be separated into two groups based on, among other criteria, serological relationships that are presumably governed by the viral capsid structure. Nucleotide sequences for the coding regions of coat proteins of approximately 22 K were identified for the MAV-PS1, P-PAV (group 1) and NY-RPV (group 2) isolates of BYDV. The MAV-PS1 and P-PAV coat protein sequences shared 71% deduced amino acid similarity whereas that of the NY-RPV isolate shared no more than 51% similarity with either the MAV-PS1 or the P-PAV sequence. Other comparisons showed that these and other BYDV coat protein sequences examined to date share a high degree of identity with those identified from other luteoviruses. Among luteovirus coat protein sequences in general, several highly conserved domains were identified whereas other domains differentiate MAV-PS1 and PAV isolates from NY-RPV and other luteoviruses. Sequence similarities and differences among BYDV coat proteins (approx. 22K) are consistent with the serological relationships exhibited by these viruses. Amino acid sequence comparisons between BYDV isolates that share common aphid vectors indicate that it is unlikely that these coat proteins are involved in aphid specificity.

Amino Acid Sequence↗

PubMed1989

Structural elements regulating zein gene expression.

Zeins are a group of alcohol-soluble proteins that are synthesized in the endosperm of developing maize seeds. These proteins are encoded by a large number of genes located on several chromosomes; based upon the number of mutants that have been isolated, zein gene regulation is complex. Comparisons of gene flanking regions reveal conserved sequences that may be important for their regulation. Studies of transformed plant tissues support the assertion that cis-acting elements with the 5' flanking regions of zein genes are required for accurate transcription. Although the genes are transcribed in transgenic tobacco and petunia plants, they are not properly regulated. This appears to be due to transcriptional effects rather than protein or mRNA instability.

Base Sequence↗

PubMed1989

The opaque-2 mutation of maize differentially reduces zein gene transcription.

Zeins, the storage proteins of maize seed, are encoded by a large multigene family that is regulated developmentally and expressed in a tissue-specific manner during endosperm development. The synthesis of these proteins is affected by mutations, such as opaque-2, that cause a reduction in the accumulation of zein proteins and mRNAs. We used nuclear run-on transcription assays to analyze the expression of zein genes in developing normal and opaque-2 endosperms and to map the origin of these transcripts with respect to the coding and noncoding regions of the genes. These analyses demonstrate that zein gene expression is regulated transcriptionally and posttranscriptionally in developing endosperm. Transcription of genes encoding alpha-zeins is inhibited significantly in opaque-2 mutants, with expression of those encoding the M(r) 22,000 proteins being almost totally blocked. Other gene subfamilies were affected but to a lesser extent.

Chromosome Mapping↗

PubMed1989

Changes in the zein composition of protein bodies during maize endosperm development.

Zeins, the seed storage proteins of maize, are synthesized during endosperm development by membrane-bound polyribosomes and transported into the lumen of the endoplasmic reticulum, where they assemble into protein bodies. To better understand the distribution of the various zeins throughout the endosperm, and within protein bodies, we used immunolocalization techniques with light and electron microscopy to study endosperm tissue at 14 days and 18 days after pollination. Protein bodies increase in size with distance from the aleurone layer of the developing endosperm; this reflects a process of cell maturation. The protein bodies within the subaleurone cell layer are the smallest and contain little or no alpha-zein; beta-zein and gamma-zein are distributed throughout these small protein bodies. The protein bodies in cells farther away from the aleurone layer are progressively larger, and immunostaining for alpha-zein occurs over locules in the central region of these protein bodies. In the interior of the largest protein bodies, the locules of alpha-zein are fused. Concomitant with the appearance of alpha-zein in the central regions of the protein bodies, most of the beta- and gamma-zeins become peripheral. These observations are consistent with a model in which specific zeins interact to assemble the storage proteins into a protein body.

Immunohistochemistry↗

PubMed1989

Analysis of avenin proteins and the expression of their mRNAs in developing oat seeds.

We have isolated and characterized cDNA clones encoding avenins, the prolamine storage proteins of oat seeds. Sequence analysis shows that avenins are a related group of polypeptides and that their mRNAs differ from each other by point mutations and small insertions and deletions. Avenin proteins have structural homology to the alpha/beta-gliadins and gamma-gliadins of wheat, the B-hordeins of barley, and the gamma-secalins of rye. Hybridization analysis of DNA from various diploid, tetraploid, and hexaploid oat species shows that the oat genome contains more globulin storage protein genes than avenin genes and that some restriction fragments containing these genes are conserved between species with common genomes. We estimate that there are 25 avenin genes and 50 globulin genes per haploid genome in Avena sativa and similar ratios of globulin to avenin genes in other Avena species. Avenin and globulin polypeptides begin to accumulate between 4 days and 6 days after anthesis. Messenger RNAs encoding avenin and globulin proteins become abundant 4 days after anthesis and reach peak concentrations at 8 days after anthesis. Avenin mRNAs are present in somewhat greater molar amounts than globulin mRNAs beginning at 4 days after anthesis. Because there is considerably more globulin than avenin in the mature oat seed, the expression of globulin and avenin genes may be regulated both transcriptionally and post-transcriptionally.

Amino Acid Sequence↗

PubMed1988

Aggregation of lysine-containing zeins into protein bodies in Xenopus oocytes.

Zeins, the storage proteins of maize, are totally lacking in the essential amino acids lysine and tryptophan. Lysine codons and lysine- and tryptophan-encoding oligonucleotides were introduced at several positions into a 19-kilodalton zein complementary DNA by oligonucleotide-mediated mutagenesis. A 450-base pair open reading frame from a simian virus 40 (SV40) coat protein was also engineered into the zein coding region. Messenger RNAs for the modified zeins were synthesized in vitro with an SP6 RNA polymerase system and injected into Xenopus laevis oocytes. The modifications did not affect the translation, signal peptide cleavage, or stability of the zeins. The ability of the modified zeins to assemble into structures similar to maize protein bodies was assayed by two criteria: assembly into membrane-bound vesicles resistant to exogenously added protease, and ability to self-aggregate into dense structures. All of the modified zeins were membrane-bound; only the one containing a 17-kilodalton SV40 protein fragment was unable to aggregate. These findings suggest that it may be possible to create high-lysine corn by genetic engineering.

Amino Acid Sequence↗

PubMed1988

Role of the 3'-poly(A) sequence in translational regulation of mRNAs in Xenopus laevis oocytes.

We investigated the effect of 3'-noncoding and poly(A) sequences on the translation and stability of membrane-bound mRNA (maize zein) and free mRNA (Xenopus beta-globin) by injecting SP6 transcripts into stage 6 Xenopus oocytes. With zein mRNA, the presence or absence of a 3'-noncoding or poly(A) sequence had little effect on mRNA stability over 24 h, and the 3'-noncoding sequence played essentially no role in mRNA translation. With short periods of incubation, e.g. 1-2 h, the presence or absence of a poly(A) tail also had little effect on zein mRNA translation; but after longer periods, translation of the poly(A-) mRNA was significantly reduced. A similar pattern of mRNA translation was observed for poly(A+) and poly(A-) Xenopus beta-globin mRNAs. These differences in translational efficiency correlated with the formation of maximally loaded polysomes (seven-eight ribosomes/mRNA) for the poly(A+) zein mRNA and a failure to form large polysomes with the poly(A-) zein mRNA. These results are consistent with a model in which the 3'-poly(A) sequence of mRNAs facilitates reinitiation of ribosomes during protein synthesis.

Animals↗

PubMed1988

Deletion of DNA sequences flanking an Mr 19,000 zein gene reduces its transcriptional activity in heterologous plant tissues.

Analysis of a series of clones containing deletions in the 5' noncoding sequence of a gene encoding an Mr 19,000 zein allowed identification of a region required for maximal transcription. Transcriptional activity was assayed in two heterologous plant systems. In one system, the Ti plasmid was used to introduce the modified zein genes into the sunflower genome. In the other system, electroporation was used to transform carrot protoplasts with plasmids containing the zein genes. For the electroporation experiments, the 5' noncoding sequences from the zein clones were linked to the protein coding sequence of chloramphenicol acetyl transferase. The results showed that an upstream sequence, delimited by nucleotides -337 and -125 with respect to the mRNA cap site, is required for maximal transcription of the gene. In contrast, very low levels of transcription were directed by constructs that contained 125 bp of 5' noncoding sequence that included the CAAT and TATA boxes, suggesting that the additional sequences (-337 to -125) further 5' exert a quantitative effect on transcription. Examination of the additional 5' sequences showed five regions that share homology with the SV40 enhancer core sequence.

Base Sequence↗