Characterization of mutations that feminize gametophytes of the fern Ceratopteris.

Gametophytes of the fern Ceratopteris are either male or hermaphroditic. Their sex is epigenetically determined by the pheromone antheridiogen, which is secreted by the hermaphrodite and induces male and represses female development in other young, sexually undetermined gametophytes. To understand how antheridiogen represses the development of female traits at the genetic level, 16 new mutations that feminize the gametophyte in the presence of antheridiogen were identified and characterized. Seven are very tightly linked to the FEM1 locus previously described. Nine others define another locus named NOTCHLESS1 (NOT1), as several of the not1 mutants lack a meristem notch. Some not1 mutations also affect sporophyte development only when homozygous, indicating that the not1 mutations are recessive and that NOT1 is also required for normal sporophyte development. The epistatic interactions among FEM1, NOT1, and other sex-determining genes are described. This information was used to expand the genetic model of the sex-determining pathway in Ceratopteris. On the basis of this model, we can say that the presence of antheridiogen leads to the activation of the FEM1 gene, which not only promotes the differentiation of male traits, but also represses female development by activating the NOT1 gene. NOT1 represses the TRA genes necessary for the development of female traits in the gametophyte.

ANI1. A sex pheromone-induced gene in ceratopteris gametophytes and its possible role in sex determination.

Antheridiogen (ACE) is a pheromone that is required for the development of male gametophytes in the homosporous fern Ceratopteris richardii. Subtractive hybridization of cDNAs isolated from ACE-treated and non-ACE-treated gametophytes was used to isolate genes that are induced by the pheromone. The expression of one gene, ANI1 (for antheridiogen induced), was induced within 3 hr of ACE treatment, but its expression was transient. Patterns of ANI1 expression in wild-type and mutant gametophytes show that ANI1 expression inversely correlates with the predicted activity of one of the sex-determining genes, TRANSFORMER5 (TRA5). These data suggest that ANI1 transcription or transcript accumulation is directly or indirectly prevented by TRA5 in the absence of ACE and that ACE inactivates the TRA5 gene or its product, leading to the upregulation of ANI1. Cycloheximide (no ACE) induced the expression of ANI1, also indicating that ANI1 expression is subject to negative regulation in the absence of ACE. The sequence and inferred protein structure of ANI1 suggest that it is a novel, extracellular protein. The secreted portion of the ANI1 protein potentially forms a beta barrel with superficial similarities to lipocalins, which bind small hydrophobic molecules such as pheromones, steroids, and odorants. ANI1 may be an extracellular carrier of ACE that is required to initiate the male program of development as the sexual fate of the young gametophyte is determined.

Characterization of homeodomain-leucine zipper genes in the fern Ceratopteris richardii and the evolution of the homeodomain-leucine zipper gene family in vascular plants.

The homeodomain-leucine zipper (HD-Zip) genes encode transcription factors that are characterized by the presence of both a homeodomain and a leucine zipper motif. They belong to the homeobox gene superfamily and have been reported only from flowering plants. This article is the first report on the ferm HD-Zip genes (named Crhb1-Crhb11) isolated from the homosporous ferm Ceratopteris richardii. Phylogenetic analyses of the II Crhb genes with previously reported angiosperm HD-Zip genes show that the Crhb genes belong to three of the four different angiosperm HD-Zip subfamilies (HD-Zip I, II, and IV), indicating that these subfamilies of HD-Zip genes originated before the diversification of the ferm and seed plant lineages. The Crhb4-Crhb8 and Crhb11 genes belong to the HD-Zip I subfamily but differ from angiosperm HD-Zip I genes by the presence of a seven-amino-acid indel in the leucine zipper motif. By the northern analyses, Crhb1 and Crhb3 were expressed only in gametophyte tissue. Expression of Crhb2 and Crhb11 genes could not be detected in any tissue examined, while all other Crhb genes were expressed in most sporophytic and gametophytic tissues. Although the functions of the Crhb genes in Ceratopteris are unknown, their patterns of expression suggest that they regulate developmental or physiological processes common to both the gametophyte and the sporophyte generations of the fern. Differences in the expression of Crhb1 between male gametophytes and male-hermaphrodite mixed populations of gametophytes suggests that the Crhb1 gene is involved in gametophytic sex determination.

Characterization of MADS homeotic genes in the fern Ceratopteris richardii.

The MADS genes encode a family of transcription factors, some of which control the identities of floral organs in flowering plants. To understand the role of MADS genes in the evolution of floral organs, five MADS genes (CMADS1, 2, 3, 4, and 6) were cloned from the fern Ceratopteris richardii, a nonflowering plant. A gene tree of partial amino acid sequences of seed plant and fern MADS genes showed that the fern genes form three subfamilies. All members of one of the fern MADS subfamilies have additional amino-terminal amino acids, which is a synapomorphic character of the AGAMOUS subfamily of the flowering plant MADS genes. Their structural similarity indicates a sister relationship between the two subfamilies. The temporal and spatial patterns of expression of the five fern MADS genes were assessed by Northern blot analyses and in situ hybridizations. CMADS1, 2, 3, and 4 are expressed similarly in the meristematic regions and primordia of sporophyte shoots and roots, as well as in reproductive structures, including sporophylls and sporangial initials, although the amount of expression in each tissue is different in each gene. CMADS6 is expressed in gametophytic tissues but not in sporophytic tissues. The lack of organ-specific expression of MADS genes in the reproductive structures of the fern sporophyte may indicate that the restriction of MADS gene expression to specific reproductive organs and the specialization of MADS gene functions as homeotic selector genes in the flowering plant lineage were important in floral organ evolution.

Sex determination in plants.

Sex determination is an important developmental event in the life cycle of all sexually reproducing plants. Recent studies of sex determination in many plant species, from ferns to maize, have been fruitful in identifying the diversity of genetic and epigenetic factors that are involved in determining the sex of the flower or individual. In those species amenable to genetic analysis, significant progress has been made toward identifying mutations that affect sex expression. By studying the interactions among these genes, pictures of how sex-determining signals are perceived to activate or repress male- or female-specific genes are emerging.

The TRANSFORMER genes of the fern Ceratopteris simultaneously promote meristem and archegonia development and repress antheridia development in the developing gametophyte.

The sex of the haploid gametophyte of the fern Ceratopteris is determined by the presence or absence of the pheromone antheridiogen, which, when present, promotes male development and represses female development of the gametophyte. Several genes involved in sex determination in Ceratopteris have been identified by mutation. In this study, the epistatic interactions among new and previously described sex-determining mutants have been characterized. These results show that sex expression is regulated by two sets of genes defined by the FEM1 and TRA loci. Each promotes the expression of either male or female traits and simultaneosly represses the expression of the other. A model describing how antheridiogen regulates the expression of these genes and the sex of the gametophyte is described. The observation that some gametophytic sex-determining mutants have phenotypic effects on the sporophyte plant indicates that sex determination in the Ceratopteris gametophyte is regulated by a mechanism that also regulates sporophyte development.

Generating autotetraploid sporophytes and their use in analyzing mutations affecting gametophyte development in the fern Ceratopteris.

The haploid gametophytes of the fern Ceratopteris richardii are autotrophic and develop independently of the diploid sporophyte plant. While haploid genetics is useful for screening and characterizing mutations affecting gametophyte development in Ceratopteris, it is difficult to assess whether a gametophytic mutation is dominant or recessive or to determine allelism by complementation analysis in a haploid organism. This report describes how apospory can be used to produce genetically marked polyploid sporophytes whose gametophyte progeny are heterozygous for mutations affecting sex determination in the gametophyte and a known recessive mutation affecting the phenotype of both the gametophyte and sporophyte. The segregation ratios of wild-type to mutant phenotypes in the gametophyte progeny of polyploid sporophyte plants indicate that all of the mutations examined are recessive. The presence of many multivalents and few univalents in meiotic chromosome preparations of spore mother cells confirm that the sporophyte plants assayed are polyploid. The DNA content of the sperm of their progeny gametophytes was also found to be approximately twice that of sperm from wild-type haploid gametophytes.

Subtractive hybridization between cDNAs from untreated and AMO-1618-treated cultures of Gibberella fujikuroi.

The gibberellin (GA) biosynthetic pathway includes four apparent cytochrome P450-mediated steps that convert kaurene to 7 alpha-hydroxykaurenoic acid. One of these reactions, the hydroxylation of kaurenoic acid to 7 alpha-hydroxykaurenoic acid, is mediated by kaurenoic acid hydroxylase. This reaction can be catalyzed in vitro by microsomal preparations from the fungus Gibberella fujikuroi (Saw.) Wr. and monitored by HPLC. Cultures grown in the presence of 84 microM AMO-1618 (an inhibitor of kaurene synthesis) had reduced levels of GA3 in fungal filtrates and decreased cell-free kaurenoic acid hydroxylase activity. However, the level of hydroxylase activity from AMO-1618-treated cultures could be induced several-fold by growing cultures in the presence of 350 microM kaurene. Since transcripts related to GA biosynthesis might be decreased in AMO-1618-treated cultures, a subtractive hybridization procedure was used to enrich cDNA fragments corresponding to messages that are more abundant in untreated than treated cultures. A fungal cDNA library was screened with the subtraction products and a clone was isolated that corresponds to two down-regulated transcripts in AMO-1618-treated cultures. This cDNA does not encode a cytochrome P450 but may be associated with GA biosynthesis.

Genetic interactions among sex-determining genes in the fern Ceratopteris richardii.

Haploid gametophytes of the fern Ceratopteris are either male or hermaphroditic. The determinant of sex type is the pheromone antheridiogen, which is secreted by the hermaphrodite and directs male development of young, sexually undetermined gametophytes. Three phenotypic classes of mutations that affect sex-determination were previously isolated and include the hermaphroditic (her), the transformer (tra) and feminization (fem) mutations. In the present study, linkage analysis and tests of epistasis among the different mutants have been performed to assess the possible interactions among these genes. The results indicate that sex determination in Ceratopteris involves at least seven interacting genes in addition to antheridiogen, the primary sex-determining signal. Two models describing how antheridiogen may influence the activity states of these genes and the sex of the gametophyte are discussed.

Evidence that the Gya, Hy and Joa antigens belong to the Dombrock blood group system.

Ten red cell samples lacking the high incidence Gya antigen were found to have the previously undescribed Do(a-b-) phenotype. Fifteen Hy- red cell samples were Do(a-b+) with weak expression of Dob and 6 Jo(a-) red cell samples were Do(a+) with weak expression of Doa. Five of the 6 Jo(a-) samples had extremely weak expression of Dob. The sixth Jo(a-) was Do(b-). Immune precipitates were prepared from radio-iodinated antigen-positive red cells with anti-Gya, -Hy, -Doa and Dob. Immunoblotting of these immune precipitates with affinity-purified anti-Gya and anti-Dob under non-reducing conditions revealed similar broadly migrating bands of M(r) 48,700-59,750, suggesting that the Doa and Dob antigens are carried on the same glycoprotein as Gya and Hy. The phenotypically associated high incidence Joa antigen has previously been shown to reside on the Gya/Hy glycoprotein. Enzyme-treated and chemically modified red cells tested with anti-Doa, -Dob, -Gya, -Hy and -Joa gave the same pattern of reactivity. We propose that Gya, Hy and Joa become part of the Dombrock blood group system and that, henceforth, the Gya/Hy-active glycoprotein be renamed the Dombrock-active glycoprotein. The Gy(a)-Hy- Jo(a-) phenotype constitutes the 'null' phenotype within the Dombrock system.

Sex-determining genes in the homosporous fern Ceratopteris.

Haploid Ceratopteris gametophytes are either hermaphroditic or male. The determinate of sex type is the pheromone antheridiogen (ACE) which is secreted by the meristic hermaphrodite and promotes ameristic male development of sexually undetermined gametophytes. Several mutations effecting the sex of the haploid gametophyte have been isolated and are described. The hermaphroditic (her) mutants are insensitive to ACE and develop as meristic hermaphrodites. These mutations effect ameristic male development in the presence of ACE but have no effect on hermaphroditic development. While most her mutations also have no effect on diploid sporophyte development, some partially ACE-insensitive her mutations have profound effects on sporophyte development. The transformer (tra) mutation effects both meristem and archegonia formation and causes the gametophyte to be an ameristic male under conditions that promote hermaphroditic development. The feminization (fem) mutation effects antheridia development in both male and hermaphroditic gametophytes and causes the gametophyte to develop as a meristic female in the absence or presence of the pheromone. The her1 tra1 double mutant is male in the absence or presence of ACE, indicating that tra1 is epistatic to her1. The phenotypes of her1, tra1 and fem1 single gene mutant phenotypes and the her1 tra1 double mutant phenotype are used to deduce a model suggesting how the products of these genes might interact in a regulatory pathway to control sex determination.

A survey of the incidence of Miltenberger antibodies among Hong Kong Chinese blood donors.

BACKGROUND: The ready availability of red cells of the Miltenberger (Mi) class III phenotype (6.28%) prompted the study of Mi antibodies among Chinese blood donors in Hong Kong, 98 percent of whom are descended from inhabitants of Guangdong Province in southern China. STUDY DESIGN AND METHODS: Red cells of the Mi class III phenotype were used to conduct a survey of the frequency of Miltenberger antibodies in 56,161 random Chinese blood donors, over a period of 12 months, using a microplate technique. RESULTS: Sera from 32 donors (0.057%) were found to contain Mi antibodies: sera from 22 contained anti-Mur + Hut; sera from 4 contained anti-Vw + Mur + Hut; sera from 4 had monospecific anti-Mur; and sera from 2 had monospecific anti-Hil. The immunoglobulin isotypes of 24 sera were mixtures of IgM and IgG, 4 were pure IgM, and 4 were pure IgG. CONCLUSION: The majority of Mi antibodies detected were naturally occurring. This survey proved useful for mass screening of random donors for the procurement of valuable Mi antisera.

Dental fluorosis and fluoride exposure in Western Australia.

Dental fluorosis in children is reported from many locations, and its prevalence may be increasing. This study aimed to measure fluorosis in 12-year-olds in fluoridated and non-fluoridated areas of Western Australia and to relate this to exposure. School dental clinics in Perth (F- 0.8 mg/L) and the Bunbury area (F- less than 0.2 mg/L) were the sampling units. Parents provided data on residence in fluoridated areas and on use of supplements and toothpaste. Exposure to water and supplemental fluoride between birth and four years was calculated. Clinical examinations (upper left central incisor) based on the TF classifications of fluorosis (which requires teeth to be dried) were conducted for 338 children in Perth and 321 in the Bunbury region. Re-examinations (n = 50) gave a weighted kappa of 0.78. Contingency analysis estimated bivariate relationships, and multiple logistic regression estimated odds ratios (OR) for risk factors. Fluorosis prevalence in the Perth region was 0.40, and in the Bunbury region 0.33 (chi 2 = 3.69, df = 1, p = 0.055). Prevalence was 0.44 in children with fluoride exposure equivalent to optimal water fluoridation and 0.20 among those with the lowest exposure (chi 2 = 35.99, df = 1, p = 0.0001). Increasing exposure was associated with higher fluorosis prevalence and higher TF scores, but overall, 27.3% of participants had TF score 1 (barely discernible), and only 9.4% had TF score greater than or equal to 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and function of the maize Spm transposable element.

The Spm's primary transcript is alternatively spliced to give at least 4 different transcripts which code for proteins with extensive overlapping domains. Two of these proteins, the tnpA and tnpD gene products, are necessary and sufficient to promote dSpm transposition in tobacco. Moreover, the tnpA gene product is also sufficient to suppress the basal level of expression observed in some dSpm-disrupted genes, and probably to activate the expression of inactive elements and of other dSpm-disrupted genes. The functions of the tnpB and tnpC gene products remain unknown. However, their structure suggests that they act as negative regulators of the Spm transposase function.

Essential large transcripts of the maize Spm transposable element are generated by alternative splicing.

We used in vitro mutagenesis and cDNA cloning to identify new Suppressor-mutator (Spm) transposable element genes. Frameshift mutations in the ORFs of the tnpA gene's first intron markedly reduce Spm activity in transgenic tobacco, indicating that intron sequences encode essential gene products. Evidence is given that Spm encodes large alternatively spliced transcripts, designated tnpB (4.9 kb), tnpC (5.7 kb), and tnpD (5.8 kb), comprising all of the tnpA exons, most of the tnpA intron 1 ORF1 sequence, and either none, part, or all of the intron 1 ORF2 sequence. Two alternative splice donor sites were identified at the end of exon 1, and the structure of the different exon 1 sequences suggests that Spm employs a novel mode of translational regulation.

Mutations, epimutations, and the developmental programming of the maize Suppressor-mutator transposable element.

Information about the structure, function and regulation of the maize Suppressor-mutator (Spm) transposable element has emerged from the genetic and molecular characterization of both deletion mutations and an unconventional type of reversible genetic change (epimutation). The element is subject to an epigenetic mechanism that can either stably inactivate it or specify one of a variety of heritable programs of differential element expression in development. The essay explores the relationship between the Spm element's epigenetic developmental programming mechanism and the determinative events central to plant development and differentiation.

Molecular mechanisms in the developmental regulation of the maize Suppressor-mutator transposable element.

The maize Suppressor-mutator (Spm) element can exist in one of three heritable forms: (1) a stably active form, (2) a stably inactive form, termed cryptic, and (3) a labile form, here termed programmable, in which the element exhibits one of a variety of heritable developmental programs of expression. Active elements are transcribed and are hypomethylated at sites upstream of the transcription start site, whereas inactive elements are transcriptionally silent and largely methylated at the upstream sites. Active (both stable and programmable), inactive programmable, and cryptic elements are unmethylated, partially methylated, and fully methylated, respectively, at sites within an 0.35-kb 80% G + C region just downstream from the transcription start site. An active Spm element in a genome with a cryptic element promotes its partial demethylation but not its transcriptional activation. In contrast, a trans-acting Spm promotes extensive demethylation and transcriptional activation of an inactive programmable element, as well as its heritable reactivation. These observations define the molecular components of the Spm element's developmental regulatory mechanism. We discuss their general relevance to the developmental regulation of gene expression.

Is the Suppressor-mutator element controlled by a basic developmental regulatory mechanism?

We report the results of genetic studies on derivatives of two different alleles of the maize a locus with an insertion of the Suppressor-mutator (Spm) transposable element in which the element is inactive, but can be reactivated readily. We present evidence that the mechanism that determines whether the element is in an active or inactive phase has two genetically distinguishable components. One determines whether or not the element is genetically active (the phase setting) and the other determines the stability of the setting in development, its heritability, and its phase in the next generation (the phase program). We show that the element's phase can be reset in a reproducible pattern during plant development. We also show that the Spm element can be reprogrammed to undergo a subsequent phase change without a concomitant phase change. The capacity to reset and reprogram the Spm element is differentially expressed within the plant in a pattern that is correlated with the developmental fate of apical and lateral meristems, suggesting the involvement of a basic developmental determination mechanism.

Working safely with ethylene oxide.

During the last few years there has been a great deal of concern about the health and safety hazards of working with Ethylene Oxide (E.O.), particularly in gas sterilisation processing. This concern is well founded. Toxicity studies in primarily animal research indicate that E.O. is a suspected cancer causing agent and possible reproductive hazard. As a result of these findings, industry, health care facilities and government have been working to reduce worker exposure to extremely low levels and thereby protect the employees health. The purpose of this paper is to discuss and recommend action plans that you can follow for employee health and protection and for regulatory compliance.

Chloroplast DNA diversity is low in a wild plant, Lupinus texensis.

Chloroplast DNA diversity was measured in an annual flowering plant, Lupinus texensis. Individual plants were collected from 21 local populations throughout the range of the species in Texas. Chloroplast DNA was isolated separately from each plant and digested with seven restriction enzymes. The most common form of the 150-kilobase-pair genome was cut at 134 sites, so that about 0.5% of the base pairs in the genome were sampled. Of the 100 plants examined, 88 had identical restriction fragment patterns. Three variant forms were found in different local populations. Two, represented in single plants, differed from wild type in the presence or absence of single restriction sites. The third variant was fixed in one of the local populations; it had lost a restriction site and also had a deletion of approximately equal to 100 base pairs. The data suggest that chloroplast DNA in this plant is much less polymorphic than mitochondrial DNA from animals and is probably less polymorphic than nuclear genes in the same plant or in animals.