Characterization of university-level introductory genetics courses in Canada.

We conducted survey research with the intent to characterize post-secondary introductory genetics (IG) education in Canada during the 1996-1997 academic year. At least a minimum data set was obtained from 47 institutions through responses to a mailed questionnaire and on-line resources. The total reported enrollment (TRE) for IG was 10,500. Over half of the TRE used one particular text. A core curriculum of topics was identified as those given more than 30 min of lecture time in at least half of reporting institutions. Slightly more than half of the TRE had laboratory exercises associated with their IG course. Laboratory exercises tended to emphasize classical transmission genetics with very few exercises in molecular genetics. For the determination of academic equivalency between institutions, particular attention should be given to the breadth and duration of the tutorial and (or) laboratory components. The majority of personnel teaching IG were trained in Canada within the previous 15 years. We suggest mechanisms by which the Genetics Society of Canada could work to promote genetical literacy.

Translation of some maize small heat shock proteins is initiated from internal in-frame AUGs.

Etiolated maize radicles (inbred Oh43) subjected to a brief heat shock synthesize a family of small heat shock proteins (approximately 18 kD) that is composed of at least 12 members. We previously described the cDNA-derived sequence of three maize shsp mRNAs (cMHSP18-1, cMHSP18-3, and cMHSP18-9). In this report, we demonstrate that the mRNA transcribed in vitro from one of these cDNAs (cMHSP18-9) is responsible for the synthesis of three members of the shsp family, and we suggest that cMHSP18-3 may be responsible for the synthesis of three additional members and cMHSP18-1 for the synthesis of two other members of this family. The fact that these genes do not contain introns, coupled with the observations reported herein, suggest that maize may have established another method of using a single gene to produce a number of different proteins.

Maize seedlings show cell-specific responses to heat shock as revealed by expression of RNA and protein.

The cellular localization of heat-shock proteins has been described in a number of experimental animal systems but is not well defined in plant systems. Sense and antisense RNA transcripts from the open reading frame (ORF) of 18-kDa maize heat-shock protein genes were employed in in situ hybridizations of inbred Oh43 radicles and plumules to reveal the locations of their mRNAs. Localization of the specific mRNAs to the younger meristematic cells of the root-tips and shoot-tips and also to the densely cytoplasmic cells of the vasculature was observed routinely. The ORF of one of our 18-kDa genes was cloned into an expression vector, and the 161-amino acid polypeptide was used to raise antibodies. Using a Fast Red procedure, the cellular positions of the heat-shock protein-antibody conjugates were observed in sections similar to those employed in the antisense RNA in situ hybridizations. The localization of the antibody appears to parallel closely the patterns of distribution of the mRNAs.

Characterization, chromosomal mapping, and expression of different ubiquitin fusion protein genes in tissues from control and heat-shocked maize seedlings.

Organisms possess at least two multigene families of ubiquitins: the polyubiquitins, with few to several repeat units, which encode a ubiquitin monomer, and the ubiquitin fusion (or extension) protein genes, which encode a single ubiquitin monomer and a specific protein. This report provides details about two ubiquitin fusion protein genes in maize referred to as MubG7 (uwo 1) and MubG10 (uwo 2). Each has one nearly identical ubiquitin coding unit fused without an intervening nucleotide to an unrelated, 237-nucleotide sequence that encodes for a 79 amino acid protein. The derived amino acid sequences of the two fusion proteins show that they differ by five amino acids (substitution by either a serine or threonine). MubG7 maps to chromosome 8L162 and MubG10 maps to chromosome 1L131. Analyses of the role(s) of these genes in response to heat shock (1 h at 42.5 degrees C) reveal that the level of these fusion protein mRNAs in the radicles or plumules from 2-day-old seedlings does not change; however, heat shock does cause a marked reduction in the accumulation of these same gene-specific mRNAs in the radicles and plumules of 5-day-old seedlings. These data confirm the suggestion from our earlier work that there is precise modulation, in a gene-specific manner, of the response to developmental as well as environmental signals.

Characterization, chromosomal mapping, and expression of different polyubiquitin genes in tissues from control and heat-shocked maize seedlings.

Polyubiquitin transcripts accumulate in plant and animal cells following a heat shock. Most species have a few to several polyubiquitin genes; within a species, the genes may differ in nucleotide (nt) sequence and (or) the number of 228-nt repeats encoding the ubiquitin monomer. This study examines three maize (inbred Oh43) polyubiquitin genes. Two of the genes, MubG9 and MubG5, possess five repeats; the third, MubG1 possesses seven repeats. Sequence analyses of the genomic clones, MubG9 and MubG1 and a cDNA clone, MubG5, reveal that they differ primarily from each other in their nt sequences 5' and 3' to their open reading frames. MubG1 contains a 1004-base pair (bp) intron in its 5' untranslated region. Using gene-specific probes, we show that the amount of polyribosome-associated mRNA transcripts from MubG9 isolated from 2- and 5-day old plumules and radicles is unchanged by heat shock. While the amount of transcript from MubG1 and MubG5 on the polyribosomes in plumules and radicles of 2-day-old seedlings is also unchanged by heat shock, the levels of these transcripts are elevated considerably in similar tissues from heat-shocked 5-day-old seedlings. Similar or identical gene-specific probes were employed to map the genes using the recombinant inbred method. MubG9 maps to chromosome 4L position 186; MubG1 maps to 5L104 and MubG5 to 4L188. The opportunity to use gene-specific probes extends the evidence for distinct modulation (time and tissue) of polyubiquitin gene expression in maize and provides the basis for locus assignment within the genome.

The independent stage-specific expression of the 18-kDa heat shock protein genes during microsporogenesis in Zea mays L.

The small (18-kDa) heat shock proteins (hsps) of maize are encoded by a complex multigene family. In a previous report, we described the genetic information from cDNAs encoding two different members of the family. In this communication, we report the isolation and characterization of cDNA and genomic clones encoding information for a third member of this hsp family (c/gMHSP18-1). DNA fragments containing nucleotide sequences common to, or specific for, each of these characterized 18-kDa genes were prepared and used as probes to assess the expression of these genes during microsporogenesis and development of the gametophyte in an inbred line of maize (Oh43). Our results demonstrate (1) that mRNA transcripts encoding the 18-kDa hsps are expressed and/or accumulate during microsporogenesis, and (2) that genes encoding two of the characterized 18-kDa hsps are expressed and/or accumulate independently, in a stage-specific manner during microsporogenesis. These observations imply that the stage-specific expression of particular 18-kDa hsp genes results from gene-specific regulation during microsporogenesis and gametophyte development rather than from an overall activation of the heat shock or stress response.

Sequence, identification and characterization of cDNAs encoding two different members of the 18 kDa heat shock family of Zea mays L.

Heat-shocked maize seedlings (cv. Oh43) synthesize a characteristic set of heat-shock proteins (hsps) which include an 18 kDa family containing at least six major isoelectric variants. A cDNA library was constructed from poly(A)+ RNAs isolated from the radicles of heat-shocked maize seedlings and screened with a DNA fragment from the theoretical open reading frame of a putative Black Mexican Sweet maize hsp18 genomic clone. Two clones, cMHSP18-3 and cMHSP18-9, were isolated, and the RNA transcripts generated from them were translated into proteins which immunoreact with antibodies directed against the maize 18 kDa hsps and exhibit the same electrophoretic characteristics as two different members of the 18 kDa hsp family. Nucleotide sequence analyses of the cDNAs in these clones reveal that their 5' and 3' untranslated regions exhibit 33-34% identity and that their protein encoding regions share 93% identity. The deduced amino acid sequences of these clones show 90% identity, and the apparent molecular masses and isoelectric points of these proteins agree with those established for two different 18 kDa hsps, numbered 3 and 6. This report substantiates that at least two of the 18 kDa hsps in maize are products of different but related genes. Moreover, it establishes that transcripts for these proteins accumulate during heat shock and that both their nucleotide and deduced amino acid sequences share extensive similarities with the class VI small hsps in soybean and with transcripts expressed during meiosis in Lilium.

Multivariate analysis of polypeptide synthesis in field-grown maize inbreds and hybrids.

A leaf disc method is described to permit the localized incorporation of (35)S-L-methionine into polypeptides synthesized in individual leaves of maize plants grown in the field. The method of incorporation employs minimal external manipulation of the intact leaf, is simple, repeatable, and may be used at any plant age after leaf emergence. Incorporation (cpm/µg protein) in 12 leaves per plant was compared among three inbred (Oh43, W23, M14) and three F1 hybrid (Oh43/M14, W23/M14, Oh43/W23) genotypes. The incorporation was 40% higher (hybrid versus inbred) in 9 of the 12 leaves studied. Samples from leaf 07 (7th leaf numbered from base of plant) for four inbreds (Oh43, M14, B73, Mol 7) and two pairs of reciprocal F1 hybrids (Oh43/M14, M14/Oh43; B73/Mo17, Mo17/B73) were labelled in situ using the leaf disc method. Each cultivar was sampled at three different ages in each of 1985, 1986, and 1987. High-resolution, two-dimensional isoelectric focusing sodium-dodecyl-sulfate polyacrylamide gel electrophoresis and fluorography were used to display the polypeptides synthesized in the samples. Multivariate methods - Principal Coordinate Analysis, Cluster Analysis, and Standard Deviation Distance - were used to analyze variation and to identify trends in the variation for year, genotype, and age sampled. Our analyses disclose a hierarchy to polypeptide synthesis variation in maize leaves: differences in polypeptide synthesis are greater for year-to-year comparisons than differences due to sample age, which in turn are greater than differences for inbred versus hybrid comparisons.

Heat Shock Protein Synthesis Induced by Methomyl in Maize (Zea mays L.) Seedlings.

Exposure of plumules of intact maize seedlings (Zea mays L.) to S-methyl-N-[(methylcarbamoyl)-oxy]thioacetimidate (methomyl) represses synthesis of several polypeptides normally made under control conditions and induces synthesis of polypeptides similar to maize heat shock polypeptides (HSPs). Three of the methomyl-induced polypeptides (18 kilodaltons) are recognized by antibodies raised against 18 kilodalton maize heat shock polypeptides.

Multivariate analyses of polypeptide synthesis in developing maize embryos.

Variation in polypeptide synthesis was examined in developing maize embryos of two inbred and two hybrid genotypes. Multivariate analyses were used to evaluate the variation among two-dimensional, electrophoretic separations of polypeptides. Several features of the data set were revealed. Similar developmental patterns were exhibited by all genotypes and no evidence was obtained for differential rates of development for inbreds and hybrids. The differential synthesis of two subsets of polypeptides during embryo development was observed. The multivariate methods employed in this study were a valuable aid in interpreting the results from a large and complex data set.

Production of normal, germinable and viable pollen from in vitro-cultured maize tassels.

Immature tassel meristems (1.0-1.5 cm long) of Zea mays L. inbred, Oh43, and single cross hybrid, Se60, cultured on a nutrient liquid medium underwent extensive development through to maturity and produced normal, mature, trinucleate pollen grains. The grains germinated on nutrient agar and on receptive silks and also produced viable kernels. No differences were observed between in vitro-produced pollen and in vivo pollen (pollen from greenhouse-grown plants) in characteristics such as pollen size, in vitro and in situ germination, and pollen tube growth in vitro. The kernels produced with in vitro pollen grew into mature plants (in vitro plants) which were similar to in vivo plants (plants produced with in vivo pollen), with no significant differences for all the morphological characteristics measured, and no phenotypic and cytological abnormalities. Gel electrophoresis of polypeptides revealed no major differences between in vitro and in vivo seedlings. This demonstration of fertilization and production of normal, uniform plants with pollen from cultured tassels has significant potential in basic and applied research studies.

Multivariate analysis of data from two-dimensional electrophoretic separation of macromolecules.

Recent work on two-dimensional electrophoretic separation of molecules has concentrated on technical aspects of molecule identification, leaving little in the way of time and resources to explore new, effective methods for analyzing the data thus produced. We present a method for coding electrophorograms that is capable of capturing sufficient information in order that subsequent analysis might be revealing. Along with this coding method, we describe a flexible approach to the analysis of electrophoretic data employing multivariate methods for summarization and comparison. In this we have found an effective vehicle for revealing some unexpected properties of the electrophorograms.

Fertilization and seed production with pollen from in-vitro-cultured maize tassels.

Immature tassel meristems (1-1.5 cm) of maize (Zea mays L.) explanted to a defined nutrient medium underwent further growth and floral development. Microsporogenesis, gametogenesis and pollen maturation were completed within 25 d in vitro. The pollen, recovered from the cultured tassel, germinated on nutrient agar and also on receptive silks. Viable seed produced from controlled pollinations germinated and grew into mature, normal plants. Thus, a significant component of the life cycle of maize can be completed in vitro where analyses and manipulations are possible for both basic and applied research.

Regulation of gene expression in corn (Zea mays L.) by heat shock. II. In vitro analysis of RNAs from heat-shocked seedlings.

Five-day-old maize seedlings subjected to heat shock exhibit a dramatic enhancement in the synthesis of a small group of polypeptides. Isolation of total RNA from control and heat-shocked maize plumules, fractionation of poly(A)+ mRNA by oligo(dT)-cellulose chromatography, and in vitro translations of the RNAs in both the rabbit reticulocyte and the wheat germ systems indicates that there is remarkable fidelity of the mRNA pool obtained from heat-shocked plumules to reproduce in vitro those same polypeptides whose synthesis is greatly elevated in the intact, heat-shocked plumule. Moreover, these heat-shock polypeptides with molecular masses of 108 000, 89 000, 84 000, 73 000, and 18 000 are translated from polyadenylated mRNAs. The absence of a 76 000 dalton heat-shock polypeptide (HSP) and the presence of fewer isoelectric point variants of the 89 000 and 84 000 dalton HSPs among the in vitro translation products suggests that translational and (or) posttranslational regulatory mechanisms might be operative in determining the final spectrum of the maize heat-shock proteins.

Regulation of gene expression in corn (Zea Mays L.) by heat shock.

Subjecting 5-day-old plumules of corn (Zea mays L.) to elevated temperatures for brief periods of time causes the pattern of protein synthesis to shift from the production of a broad spectrum of proteins to the new and (or) enhanced synthesis of a small number of heat-shock polypeptides (HSPs). Most notable is the depressed synthesis of a major polypeptide (relative mass (Mr) = 93 000 and isoelectric point = 8.0) normally made at 27 degrees C and the enhanced and (or) new synthesis of polypeptides with MrS of 108 000, 89 000, 84 000, 76 000, 73 000, and 18 000, following 1 h of heat shock. These six HSPs is observed within 120 min following heat shock. Recovery from heat shock is rapid; after 6 to 8 h at 27 degrees C following heat shock, the polypeptide pattern is indistinguishable from the control. Extracts from individual heat-shocked shoots produced polypeptide synthetic patterns identical to those from extracts from 20 shoots, regardless of whether single shoots were intact or excised during labelling. Single 5-day-old primary roots exhibited polypeptide synthetic patterns and responded to heat shock in a manner similar to shoots. This is the first demonstration of the induction of heat-shock polypeptides in a whole, intact higher plant.

Affinity Distance Values among Somatic Metaphase Chromosomes in Maize.

In maize root-tip metaphase preparations, all distances between two chromosomes were measured in 50 cells from each of seven stocks and in 30 from one stock; four were arrested with cold, two with 8-hydroxyquinoline, one with colchicine and one with monobromonaphthalene. Standardized, affinity-distance values were calculated for all pairs of homologues and pairs of nonhomologues from each preparation. The homologues of pair X were the least separated, those of pair I the most separated in the cold-arrested stocks. All but pairs I and VIII were shown to be significantly different from the observed mean. The observed mean was less than but not significantly different from the theoretical value for a random distribution. The use of chemical agents for metaphase arrest increased the separation of homologues, except for pair I.-Eleven percent of the comparisons of nonhomologues from cold-arrested, as contrasted to none of the comparisons from the c-metaphase treatments, were significantly different from the theoretical value for a random distribution. This was considered evidence for limited primary nonhomologue association in maize. Although there were specific, differential responses to the two arrest agents, the population of homologous pairs approached a random distribution only in chemically arrested stocks.-Primary homologue association was considered to be maintained by two mechanisms, the more common involving the microtubules and the second involving the nucleolus.-Interpretations are offered regarding the claims of somatic association in other species, especially man. The opportunity in maize for experimentally modifying distance values by cytogenetic techniques is discussed.