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Triticum aestivum puroindolines, two basic cystine-rich seed proteins: cDNA sequence analysis and developmental gene expression.

From a mid-maturation seed cDNA library we have isolated cDNA clones encoding two Triticum aestivum puroindolines. Puroindoline-a and puroindoline-b, which are 55% similar, are basic, cystine-rich and tryptophan-rich proteins. Puroindolines are synthesized as preproproteins which include N- and C-terminal propeptides which could be involved in their vacuolar localization. The mature proteins have a molecular mass of 13 kDa and a calculated isoelectric point greater than 10. A notable feature of the primary structure of puroindolines is the presence of a tryptophan-rich domain which also contains basic residues. A similar tryptophan-rich domain was found within an oat seed protein and a mammalian antimicrobial peptide. The ten cysteine residues of puroindolines are organized in a cysteine skeleton which shows similarity to the cysteine skeleton of other wheat seed cystine-rich proteins. Northern blot analysis showed that puroindoline genes are specifically expressed in T. aestivum developing seeds. No puroindoline transcripts as well as no related genes were detected in Triticum durum. The identity of puroindolines to wheat starch-granule associated proteins is discussed as well as the potential role of puroindolines in the plant defence mechanism.

Amino Acid Sequence↗

A novel regulation on developmental gene expression of fruiting body formation in Myxobacteria.

Myxobacteria are Gram-negative soil microorganisms that prey on other microorganisms. Myxobacteria have significant potential for applications in biotechnology because of their extraordinary ability to produce natural products such as secondary metabolites. Myxobacteria also stand out as model organisms for the study of cell-cell interactions and multicellular development during their complex life cycle. Cellular morphogenesis during multicellular development in myxobacteria is very similar to that in the eukaryotic soil amoebae. Recent studies have started uncovering molecular mechanisms directing the myxobacterial life cycle. We describe recent studies on signal transduction and gene expression during multicellular development in the myxobacterium Myxococcus xanthus. We provide our current model for signal transduction pathways mediated by a two-component His-Asp phosphorelay system and a Ser/Thr kinase cascade.

Gene Expression Regulation, Bacterial↗

Neuronatin mRNA: alternatively spliced forms of a novel brain-specific mammalian developmental gene.

Neurogenesis begins with the closure of the neural tube around mid gestation and continues in the rat for about two weeks postnatally. Therefore, we investigated the role of neuronatin, a novel cDNA that we cloned from neonatal rat brain (Joseph et al., Biochem. Biophys. Res. Commun., 201 (1994) 1227-1234), in brain development. Further studies described in the present manuscript, lead to the identification of two alternatively spliced forms of neuronatin mRNA, alpha and beta, with the same open reading frame. Neuronatin-alpha encoded a novel protein of 81 aa, and the beta-form encoded 54 aa. Both forms were identical, except that the alpha-form had an additional 81 bp sequence inserted into the middle of the coding region. On Northern analyses, neuronatin mRNA was relatively selective for the brain. It first appeared at E11-14, a time when the neural tube has closed and neuroepithelial proliferation initiated, became pronounced at E16-19 with a surge in neurogenesis, and declined postnatally to adult levels with the completion of neurogenesis. In order to determine whether there were other forms of neuronatin mRNA, and to study the expression of the alpha and beta forms separately during development, reverse transcriptase-polymerase chain reaction was carried out using primers flanking the coding region of the alpha and beta forms. The RT-PCR results clearly indicated that there were only two forms of neuronatin. The beta-form first appeared at E11-14, whereas the alpha-form was present even earlier at E7-10. Together, these findings indicate that the two forms of neuronatin mRNA are regulated differently during brain development.(ABSTRACT TRUNCATED AT 250 WORDS)

Alternative Splicing↗

Structure and evolution of goat gamma-, beta C- and beta A-globin genes: three developmentally regulated genes contain inserted elements.

We have determined the complete nucleotide sequence of the goat fetal (gamma), preadult (beta C) and adult (beta A) globin genes. In contrast to other globin genes expressed in different stages of development, these three genes are highly homologous (approximately 90%) in both their coding and noncoding regions. The only major difference between them results from elements inserted into their large introns: gamma contains a 247 bp insertion, beta A contains a 318 bp insertion, and beta C contains both a 252 bp insertion and a 60 bp deletion. Based on comparisons of these three genes to each other and to two goat globin pseudogenes, psi beta X and psi beta Z, we conclude that gamma, beta A and beta C have diverged relatively recently, and that the two gene pairs psi beta X - beta C and psi beta Z-beta A arose via a block duplication of an ancestral pseudogene-functional gene pair. Moreover, as opposed to the human fetal genes (G gamma and A gamma), the goat gamma gene appears to be a true beta-like gene that has fetal-specific function. The insertions in gamma, beta A and beta C contain sequence features characteristic of transposon-like elements, and appear to be represented in multiple copies throughout the genome.

Animals↗

Membrane sites regulating developmental gene expression in Dictyostelium discoideum.

Postaggregative gene expression in Dictyostelium discoideum requires cell contact. Polyspecific monovalent antibodies (Fab) prepared from sera raised against membranes of aggregation- and postaggregation-stage cells were used to probe the cell interactions that induce rapid postaggregative synthesis of UDP-glucose pyrophosphorylase. When cells of strain V12M2 were dissociated after 8 hr of development and replated in the presence of immune Fab, both reaggregation and pyrophosphorylase synthesis were blocked. Fab neutralized by incubation with EDTA-high salt extracts of cells developed for 3 hr blocked pyrophosphorylase synthesis but not reaggregation. Therefore, some cell-surface components that regulate pyrophosphorylase synthesis (called E sites) are antigenically distinct from those required for reaggregation. The Fab provides a means to assay E sites during their purification. Addition of 10(-3) M cyclic AMP or cyclic GMP enabled the cells to bypass the blocking of E sites by Fab; pyrophosphorylase was synthesized in the absence of reaggregation. We hypothesize that E sites function by raising the level of intracellular cyclic AMP.

Cell Communication↗

The Viviparous-1 developmental gene of maize encodes a novel transcriptional activator.

The Viviparous-1 (Vp1) gene of maize is specifically required for expression of the maturation program in seed development. We show that Vp1 encodes a 73,335 dalton protein with no detectable homology to known proteins. An acidic transcriptional activation sequence was identified by fusion to the GAL4 DNA-binding domain. Expression of VP1 in maize protoplasts resulted in strong activation (greater than 130-fold) of a reporter gene fused to the promoter of a presumptive target gene. The acidic domain in VP1 was essential for transactivation and could be functionally replaced by the activator sequence of the herpes simplex virus VP16 protein. Our results indicate that VP1 is a novel transcription factor possibly involved in potentiation of a seed-specific hormone response.

Amino Acid Sequence↗

Developmental gene expression of a 230-kilodalton macrogamete-specific protein of the avian coccidial parasite, Eimeria maxima.

We prepared a cDNA library from gametocytes of Eimeria maxima and screened it using antibodies raised against an 82-kDa gametocyte antigen. One cDNA clone designated pEM230 was isolated and characterized. It encodes a portion of a 230-kDa gametocyte protein and its DNA sequence shows the presence of several tandem repeats of 42 bp. In order to determine the stage and sex specificity of the mRNA for the 230-kDa protein, Northern blotting and in situ hybridization studies were performed. The 230-kDa protein is encoded for by a 7 kb mRNA, which is expressed exclusively during the macrogamete stage with no detectable expression seen in any other stage of parasite development.

Amino Acid Sequence↗

The Drosophila melanogaster developmental gene g1 encodes a variant zinc-finger-motif protein.

In Drosophila melanogaster, the mechanisms involved in the pattern formation of complex internal organs are still largely unknown. However, the identity of the molecular determinants that control the development of these specific tissues is emerging from the combined use of genetic and molecular approaches. We have cloned a gene that is expressed in the mesoderm, one of the fundamental embryonic germ layers which gives rise to internal structures, such as the musculature. Here, we describe the molecular characterization of this gene, designated as g1. The nucleotide (nt) sequence of its cDNA shows an open reading frame of 852 nt, which encodes a 32-kDa protein with two putative zinc fingers, and a serine/glutamine/proline-rich region. These features indicate a functional role for g1, which remains to be elucidated, in regulating gene expression during mesoderm formation.

Amino Acid Sequence↗

Pax-2: a developmental gene constitutively expressed in the mouse epididymis and ductus deferens.

PURPOSE: To characterize expression of the transcriptional activator, Pax-2, in the mouse lower genitourinary tract. MATERIALS AND METHODS: Expression of Pax-2 was studied by Northern analysis, ribonuclease protection and immunohistochemistry. RESULTS: Immunostaining revealed localized expression in the epithelium of the ductus deferens and epididymis at all time points from birth to adulthood. Expression in these structures in adult mice was confirmed by Northern analysis and ribonuclease protection assays. CONCLUSION: Pax-2 is a transcriptional regulator expressed in the epithelium of the ductus deferens and epididymis and may be a regulator of epithelial genes involved in sperm maturation and support.

Animals↗

The developmental gene Knotted-1 is a member of a maize homeobox gene family.

The Knotted-1 (Kn1) locus is defined by several dominant gain-of-function mutations that alter leaf development. Foci of cells along the lateral veins do not differentiate properly, but continue to divide, forming outpocketings or knots. The ligule, a fringe normally found at the junction of leaf blade and sheath, is often displaced and perpendicular to its normal position. The phenotype is manifested in all cell layers of the leaf blade, but is controlled by a subgroup of cells of the inner layer. Mutations result from the insertion of transposable elements or a tandem duplication. We show that the Kn1 gene encodes a homeodomain-containing protein, the first identified in the plant kingdom. Sequence comparisons strongly suggest that Kn1 acts as a transcription factor. Here we use the Kn1 homeobox to isolate other expressed homeobox genes in maize. The Kn1 homeobox may permit the isolation of genes that, like animal and fungal counterparts, regulate cell fate determination.

Amino Acid Sequence↗

Specific developmental gene silencing in the honey bee using a homeobox motif.

Manipulating the expression of genes in species that are not currently used as genetic models will provide comparative insights into the evolution of gene functions. However the experimental tools in doing so are limited in species that have not served as models for genetic studies. We have examined the effects of double stranded RNA (dsRNA) in the honey bee, an insect with considerably basic scientific interest. dsRNA derived from a 300 bp stretch of the E30 homeobox motif was injected into honey bee embryos at the anterior pole in the preblastoderm stage. We found that the dsRNA fragment successfully disrupted the protein expression of the target gene throughout the whole embryo. The disruption caused deficient phenotypes similar to known loss of function mutants of Drosophila engrailed, whereas embryos injected with nonsense dsRNA showed no abnormalities. We show that the large size of the honey bee egg (D: 0.3 mm, L: 1.6 mm) and the long preblastoderm stage (11-12 h) can be exploited to generate embryos with partial disruption of gene function, which may provide an elegant alternative to classical chimeric analyses. This is the first report of targeted disruption of gene function in the honey bee, and the results prove that the chosen target gene is a functional ortholog to engrailed in Drosophila.

Animals↗