D. Peter Snustad

Differential expression of six glutamine synthetase genes in Zea mays

The maize genome has been shown to contain six glutamine synthetase (GS) genes with at least four different expression patterns. Noncoding 3′ gene-specific probes were constructed from all six GS cDNA clones and used to examine transcript levels in selected organs by RNA gel blot hybridization experiments. The transcript of the single putative chloroplastic GS2 gene was found to accumulate primarily in green tissues, whereas the transcripts of the five putative GS1 genes were shown to accumulate preferentially in roots. The specific patterns of transcript accumulation were quite distinct for the five GS1 genes, with the exception of two closely related genes.

Dominance interactions in Escherichia coli cells mixedly infected with bacteriophage T4D wild-type and amber mutants and their possible implications as to type of gene-product function: Catalytic vs. stoichiometric

Abstract Degrees of dominance of wild-type over amber alleles have been investigated for many genes of phage T4 by measuring progeny phage burst sizes of restrictive host cells mixedly infected with varying doses of each allele. The data presented demonstrate that whereas certain am mutants are almost completely recessive to wild-type others show considerable codominance with wild type. The results suggest that the expression of a single wild-type gene specifying an enzymatic protein is likely to be sufficient for a normal burst despite gene-product interactions. The expression of a single gene specifying a structural protein, on the other hand, appears generally not to be sufficient to yield a normal burst size. Applied to am mutants in genes controlling unknown functions involved in T4 morphogenesis, this criterion suggests that genes 4, 26, 28, 31, 38, 50, 51, and 65 are the most likely to code for gene products which function catalytically (if, in fact, any such functions exist).

α-Tubulin gene family of maize (Zea mays L.) : evidence for two ancient α-tubulin genes in plants

Abstract Among 81 α-tubulin cDNA clones prepared from RNA from maize seedling shoot, endosperm and pollen, we identified six different α-tubulin coding sequences. The DNA sequence analysis of coding and non-coding regions from the clones showed that they can be classified into three different α-tubulin gene subfamilies. Genes within each subfamily encode proteins that are 99 to 100% identical in amino acid sequence. Deduced amino acid sequence identity between genes in different subfamilies ranges from 89 to 93 %. The results of hybridizations of genomic DNAs to α-tubulin coding region probes and to 3′ non-coding region probes constructed from six different α-tubulin cDNA clones indicated that the maize α-tubulin gene family contains at least eight members. Comparison of deduced α-tubulin amino acid sequences from maize and the dicot species Arabidopsis thaliana showed that α-tubulin isotypes encoded by genes in maize subfamilies I and II are more similar to specific Arabidopsis gene products (96 to 97% amino acid identity) than to isotypes encoded by genes in the other maize subfamilies. Phylogenetic analyses revealed that genes in these two subfamilies were derived from two ancient α-tubulin genes that predate the divergence of monocots and dicots. These same analyses revealed that the gene in maize subfamily III is more closely related to sequences from subfamily I genes than to those from subfamily IT genes. However, the subfamily III gene has no close counterpart in Arabidopsis. We found evidence of a transposable element-like insertion in the subfamily III gene in some maize lines.

The α1-tubulin gene of Arabidopsis thaliana: primary structure and preferential expression in flowers.

The primary structure of the α1-tubulin gene of Arabidopsis thaliana was determined and the 5′ and 3′ ends of its transcript were identified by S1 nuclease mapping experiments. The information obtained was used to (i) predict the amino acid sequence of the α1-tubulin, (ii) deduce the positions of introns within the α1-tubulin gene, and (iii) construct 3′ noncoding gene-specific hybridization probes with which to study the pattern of α1-tubulin transcript accumulation in different tissues and at different stages of development. The predicted amino acid sequence of the α1-tubulin has 92% identity with the predicted product of the previously characterized A. thaliana α3-tubulin gene. The coding sequence of the α1-tubulin gene is interrupted by four introns located at positions identical to those of the four introns in the α3 gene. RNA blot hybridization studies carried out with an α1-tubulin gene-specific probe showed that the α1 gene transcript accumulates primarily in flowers, with little transcript present in RNA isolated from roots or leaves. In order to investigate the pattern of α-tubulin gene expression in developing flowers, RNA was isolated from flowers at five different stages of development: flower buds, unopened flowers with pollen, open flowers, flowers with elongating carpels, and green seed pods. RNA blot hybridizations performed with 3′ noncoding gene-specific probes showed that the α3 tubulin gene transcript is present in flowers at all stages of development, whereas the α1-tubulin gene transcript could only be detected in RNA from unopened flowers with pollen, open flowers, and flowers with elongating carpels.

Characterization of four new β-tubulin genes and their expression during male flower development in maize (Zea mays L.)

Four different β-tubulin coding sequences were isolated from a cDNA library prepared from RNA from maize seedling shoots. The four genes (designated tub4, tub6, tub7 and tub8) represented by these cDNA clones together with the tub1 and tub2 genes reported previously encode six β-tubulin isotypes with 90–97.5% amino acid sequence identity. Results from phylogenetic analysis of 17 β-tubulin genes from monocot and dicot plant species indicated that multiple extant lines of β-tubulin genes diverged from a single precursor after the appearance of the two major subfamilies of α-tubulin genes described previously. Hybridization probes from the 3′ non-coding regions of six β-tubulin clones were used to quantify the levels of corresponding tubulin transcripts in different maize tissues including developing anthers and pollen. The results from these dot blot hybridization experiments showed that all of the β-tubulin genes were expressed in most tissues examined, although each gene showed a unique pattern of differential transcript accumulation. The tub1 gene showed a high level of transcript accumulation in meristematic tissues and almost no accumulation in the late stages of anther development and in pollen. In contrast, the level of tub4 transcripts was very low during early stages of male flower development but increased markedly (more than 100 times) during the development of anthers and in pollen. Results from RNAse protection assays showed that this increased hybridization signal resulted from expression of transcripts from one or two genes closely related to tub4. The tub4-related transcripts were not present in shoot tissue. Transcripts from the tub2 gene accumulated to very low levels in all tissues examined, but reached the highest levels in young anthers containing microspore mother cells. RNAse protection assays were used to measure the absolute levels of α- and β-tubulin transcripts in seedling shoot and in pollen. The α-tubulin gene subfamily I genes (tua1, tua2, tua4) contributed the great majority of α-tubulin transcripts in both shoot and pollen. Transcripts from the β-tubulin genes tub4, tub6, tub7, and tub8 were predominant in shoot, but were much less significant than the tub4-related transcripts in pollen.

Root- and shoot-specific responses of individual glutamine synthetase genes of maize to nitrate and ammonium

The responses of the five cytosolic-type glutamine synthetase (GS1) genes of maize to treatment of hydroponically grown seedlings with 10 mM KNO3 or 10 mM NH4Cl were analyzed. Non-coding 3′ gene-specific hybridization probes and radioanalytic imaging were used to quantitate individual gene transcript levels in excised roots and shoots before treatment and at selected times after treatment. Genes GS1−1 and GS1−2 exhibited distinct organ-specific responses to treatment with either nitrogen source. The GS1−1 transcript level increased over three-fold in roots, but changed little if any in shoots. In contrast, the GS1−2 transcript level increased over two-fold in shoots, but decreased in roots after treatment. Increased transcript levels were evident at 4 h after treatment with either nitrogen source, with maximum accumulations present at 8 h after treatment with ammonium and at 10–12 h after treatment with nitrate. The GS1−3 gene transcript level showed little or no change after treatment with either nitrogen source. The GS1−4 gene transcript level remained constant in shoots of treated seedlings, whereas in roots, it exhibited relatively minor, but complex responses to these two nitrogen sources. The GS1−5 gene transcript is present in very small amounts in seedlings, making it difficult to analyze its response to metabolites in young plants. These results provide support for the possibility that different cytosolic GS genes of maize play distinct roles in nitrogen metabolism during plant growth and differentiation.

Semi-constitutive expression of an Arabidopsis thaliana α-tubulin gene

In Arabidopsis tissues, the pool of tubulin protein is provided by the expression of multiple α-tubulin and β-tubulin genes. Previous evidence suggested that the TUA2 α-tubulin gene was expressed in all organs of mature plants. We now report a more detailed analysis of TUA2 expression during plant development. Chimeric genes containing TUA2 5′-flanking DNA fused to the β-glucuronidase (GUS) coding region were used to create transgenic Arabidopsis plants. Second-generation progeny of regenerated plants were analyzed by histochemical assay to localize GUS expression. GUS activity was seen throughout plant development and in nearly all tissues. The blue product of GUS activity accumulated to the highest levels in tissues with actively dividing and elongating cells. GUS activity was not detected in a few plant tissues, suggesting that, though widely expressed, the TUA2 promoter is not constitutively active.

Temporal and spatial expression patterns of TUB9, a β-tubulin gene of Arabidopsis thaliana

Transgenic plants carrying chimeric genes composed of segments of the 5′-flanking region of the Arabidopsis 9-tubulin gene (TUB9) fused to the coding region of the β-glucuronidase (GUS) gene of Escherichia coli were used to investigate the temporal and spatial patterns of TUB9 expression. Chimeric genes that contained at least 800 bp of TUB9 5′-flanking DNA were expressed primarily in floral tissues, with high levels of expression observed in pollen, elongating pollen tubes and ovules. The expression of the reporter genes in ovules ceased at the time of fertilization. In situ hybridization was used to verify that the reporter gene expression in pollen of transgenic plants is representative of the patterns of expression of the endogenous TUB9 gene. In situ hybridization also provided new insight into TUB9 transcript accumulation in ovules. The possible role of TUB9 and the functional implication of the largely non-overlapping expression patterns of tubulin genes are discussed.

DNA synthesis in bacteriophage T4-infected Escherichia coli: Evidence supporting a stoichiometric role for gene 32-product☆

Abstract When amber-restrictive Escherichia coli cells were multiply infected with T4 bacteriophage carrying varying doses of am and am+ alleles of gene 32, the average burst size was found to vary with the am+ to am input ratio. As the am+ to am input ratio was decreased, the burst size decreased sharply from that observed in am+ -infected cells. This result was observed with four independently isolated gene 32 am mutants. The decrease in burst size appears to result directly from a decrease in the rate of phage DNA synthesis since the latter was found to decrease rapidly as the gene 32 am+ to am input ratio was decreased. These results suggest that gene 32 protein may play a structural role in phage DNA metabolism.

Mutants of bacteriophage T4 deficient in the ability to induce nuclear disruption: I. Isolation and genetic characterization☆

Nuclear disruption in T4 phage-infected Escherichia coli as well as the morphology of the nuclear regions in uninfected E. coli can be observed by phase microscopy of cells spread on a thin layer of 17.5% gelatin. We have used this procedure to identify for the first time mutants of phage T4 which fail to induce nuclear disruption. The mutant phenotypes have been further characterized by thin-section electron microscopy. Nuclear disruption is not essential for phage growth. Burst-size and growth-rate experiments indicate that the nuclear disruption-deficient (ndd) mutants grow as well as wild-type T4D under the conditions and in the E. coli strains commonly used in our laboratory. Mapping experiments using multiple amber mutants and rII mutants with deletions extending into the D region adjacent to the rIIB gene indicate that the ndd mutations are located in gene D2b.