A second L-type isozyme of potato glucan phosphorylase: cloning,antisense inhibition and expression analysis

In potato tubers two starch phosphorylase isozymes, types L and H, have been described and are believed to be responsible for the complete starch breakdown in this tissue. Type L has been localized in amyloplasts, whereas type H is located within the cytosol. In order to investigate whether the same isozymes are also present in potato leaf tissue a cDNA expression library from potato leaves was screened using a monoclonal antibody recognizing both isozyme forms. Besides the already described tuber L-type isozyme a cDNA clone encoding a second L-type isozyme was isolated. The 3171 nucleotide long cDNA clone contains an uninterrupted open reading frame of 2922 nucleotides which encodes a polypeptide of 974 amino acids. Sequence comparison between both L-type isozymes on the amino acid level showed that the polypeptides are highly homologous to each other, reaching 81–84% identity over most parts of the polypeptide. However the regions containing the transit peptide (amino acids 1–81) and the insertion sequence (amino acids 463–570) are highly diverse, reaching identities of only 22.0% and 29.0% respectively.Northern analysis revealed that both forms are differentially expressed. The steady-state mRNA levels of the tuber L-type isozyme accumulates strongly in potato tubers and only weakly in leaf tissues, whereas the mRNA of the leaf L-type isozyme accumulates in both tissues to the same extent. Constitutive expression of an antisense RNA specific for the leaf L-type gene resulted in a strong reduction of starch phosphorylase L-type activity in leaf tissue, but had only sparse effects in potato tuber tissues. Determination of the leaf starch content revealed that antisense repression of the starch phosphorylase activity has no significant influence on starch accumulation in leaves of transgenic potato plants. This result indicated that different L-type genes are responsible for the starch phosphorylase activity in different tissues, but the function of the different enzymes remains unclear.     

Inheritance of gusA and neo genes in transgenic rice

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T₀) transformants and their progeny plants. T₀ plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T₁ to T₂ plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T₂ generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.     

Promoters from kin1 and cor6.6, two Arabidopsis thaliana low-temperature-and ABA-inducible genes,direct strong β-glucuronidase expression in guard cells,pollen and young developing seeds

The ability of most higher plants to withstand freezing can be enhanced by cold acclimation, although the freezing tolerance of plant tissues is also affected by their developmental stage. In addition, low temperature has pleiotropic effects on many plant developmental processes such as vernalization. The interaction between plant development and low temperature implies that some genes are regulated by both environmental factors and developmental cues. Although a number of cold-inducible genes from plants have been identified, information concerning their regulation during plant development is limited. In order to understand their developmental regulation and obtain possible clues as to function, the promoters of kin1 and cor6.6, two cold- and abscisic acid (ABA)-regulated genes from Arabidopsis thaliana, were fused to the -glucuronidase (GUS)-coding sequence and the resulting constructs were used to transform tobacco and A. thaliana. Transgenic plants with either the kin1 or cor6.6 promoter showed strong GUS expression in pollen, developing seeds, trichomes and, most interestingly, in guard cells. During pollen development, maximum GUS activity was found in mature pollen. In contrast, the maximum GUS activity during seed development was during early embryogenesis. These patterns of expression distinguish kin1 and cor6.6 from related lea genes which are strongly expressed during late embryogenesis. There was no major qualitative difference in patterns of GUS expression between kin1 and cor6.6 promoters and the results were similar for transgenic tobacco and Arabidopsis. Considering the results described, as well as those in an accompanying paper Wang et al., 1995, Plant Mol Biol 28: 605–617 (this issue), we suggest that osmotic potential might be a major factor in regulating the expression of kin1 and cor6.6 during several developmental processes. The implication of the results for possible function of the gene products is discussed.     

Definition of constitutive gene expression in plants: the translation initiation factor 4A gene as a model

The NeIF-4A10 gene belongs to a family of at least ten genes, all of which encode closely related isoforms of translation initiation factor 4A. The promoter region of NeIF-4A10 was sequenced, and four mRNA 5 ends were determined. Deletions containing 2750, 689 and 188 bp of untranscribed upstream DNA were fused to the GUS reporter gene and introduced into transgenic tobacco. The three constructs mediated GUS expression in all cells of the leaf, stem and shoot apical meristem. Control experiments using in situ hybridization and tissue printing indicated that the observed GUS expression matches the expression patterns of NeIF-4A mRNA and protein. This detailed analysis at the level of mRNA, protein and reporter gene expression shows that NeIF-4A10 is an ideal constitutively expressed control gene. We argue that inclusion of such a control gene in experiments dealing with specifically expressed genes is in many cases essential for the correct interpretation of observed expression patterns.     

Islands: stability, diversity, conservation

Islands present both a diversity and a stability paradox. They are often highly species-poor but have considerable biological interest in terms of extraordinary endemic genera and taxonomically isolated groups. They appear to be stable, as in some cases these organisms have persisted for many millions of years, and having an oceanic climate, extreme climatic events may be comparatively rare. However, when subject to extrinsic (anthropogenic) disturbance they do not appear to be stable, but often suffer catastrophic ecological change. These apparent paradoxes are resolved when it is realized that all these features are consequences of the same island characteristics: biotic isolation and oceanicity. As a result of these two characteristics, far oceanic islands are quantitatively different from continental systems in the nature of their ecological processes, which appear to give rise to an extreme punctuated equilibrium model of evolutionary change. Endemics may be ancient relict endemics displaying prolonged stasis and persistence, or products of adaptive radiation representing rapid punctuational events. A process-based definition of a relict endemic (palaeoendemic) is one whose founding lineage (i.e. the original continental source taxon) has not left any descendents. A corollary of this definition is that the time of divergence between an endemic and its continental sister-group should predate the colonization of the island by the now endemic lineage. An example is Dicksonia arborescens which has been on St Helena for at least 9 Myrs and no longer occurs in the likely source area of Africa. These relict endemics, frequent on islands, are important as the last remnants of tranches of biodiversity that have vanished elsewhere. Island conservation strategies require an integrated understanding of both sides of the diversity and stability paradox so that both island processes and island organisms can be conserved.     

Tissue-specific expression in transgenic maize of four endosperm promoters from maize and rice

The tissue-specific, developmental, and genetic control of four endosperm-active genes was studied via expression of GUS reporter genes in transgenic maize plants. The transgenes included promoters from the maize granule-bound starch synthase (Waxy) gene (zmGBS), a maize 27 kDa zein gene (zmZ27), a rice small subunit ADP-glucose pyrophosphorylase gene (osAGP) and the rice glutelin 1 gene (osGT1). Most plants had a transgene expression profile similar to that of the endogenous gene: expression in the pollen and endosperm for the zmGBS transgene, and endosperm only for the others. Histological analysis indicated expression initiated at the periphery of the endosperm for zmGBS, zmZ27 and osGT1, while osAGP transgene activity tended to start in the lower portion of the seed. Transgene expression at the RNA level was proportional to GUS activity, and did not influence endogenous gene expression. Genetic analysis showed that there was a positive dosage response with most lines. Activity of the zmGBS transgene was threefold higher in a low starch (shrunken2) genetic background. This effect was not seen with zmZ27 or osGT1 transgenes. The expression of the transgenes is discussed relative to the known behaviour of the endogenous genes, and the developmental programme of the maize endosperm     

Tolerance to the fungal pathogen Rhizoctonia solani AG4 of transgenic tobacco expressing the maize ribosome-inactivating protein b-32

The maize b-32 protein is a functional ribosome-inactivating protein (RIP), inhibiting in vitro translation in the cell-free reticulocyte-derived system and having specific N-glycosidase activity on 28S rRNA. Previous results indicated that opaque-2 (o2) mutant kernels, lacking b-32, show an increased susceptibility to fungal attack and insect feeding and that ectopic expression in plants of a barley and a pokeweed RIP leads to increased tolerance to fungal and viral infection. This prompted us to test whether b-32 might functi on as a protectant against pathogens. The b32.66 cDNA clone under the control of the potato wun1 gene promoter was introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Out of 23 kanamycin resistant regenerated shoots, 16 contained a PCR fragment of the corrrect size spanning the boundary between the promoter used and the coding region of the b-32 gene. Eight independently transformed tobacco lines were randomly chosen for protein analysis: all of them expressed b-32 protein. The data presented indicate that transgenic tobacco plants expressing b-32 show an increased tolerance against infection by the soil-borne fungal pathogen Rhizoctonia solani Kuhn     

Actions and interactions of growth hormone and insulin-like growth factor-II: body and organ growth of transgenic mice

To characterize long-term actions and interactions of growth hormone (GH) and insulin-like growth factor-II (IGF-II) on postnatal body and organ growth, hemizygous phosphoenolpyruvate carboxykinase (PEPCK)-human IGF-II transgenic mice were crossed with hemizygous PEPCK-bovine GH transgenic mice. The latter are characterized by two-fold increased serum levels of IGF-I and exhibit markedly increased body, skeletal and organ growth. Four different genetic groups were obtained: mice harbouring the IGF-II transgene (I), the bGH transgene (B), or both transgenes (IB), and non- transgenic controls (C). These groups of mice have previously been studied for circulating IGF-I levels (Wolf et al., 1995a), whereas the present study deals with body and organ growth. Growth curves (week 3 to 12) were estimated by regression with linear and quadratic components of age on body weight and exhibited significantly (p < 0.001) greater linear coefficients in B and IB than in I and C mice. The linear coefficients of male I and C mice were significantly (p < 0.001) greater than those of their female counterparts, whereas this sex-related difference was absent in the bGH transgenic groups. The weights of internal organs as well as the weights of abdominal fat, skin and carcass were recorded from 3.5- to 8- month-old mice. In addition, organ weight-to-body weight-ratios (relative organ weights) were calculated. Except for the weight of abdominal fat, absolute organ weights were as a rule significantly greater in B and IB than in I and C mice. IGF-II overproduction as a tendency increased the weights of kidneys, adrenal glands, pancreas and uterus both in the absence and presence of the bGH transgene. Analysis of relative organ weights demonstrated significant (p < 0.05) effects of elevated IGF- II on the relative growth of kidneys (males and females) and adrenal glands (females), confirming our previous report on organ growth of PEPCK-IGF-II transgenic mice. In females, IGF-II and GH overproduction were additive in stimulating the growth of spleen and uterus, providing evidence for tissue-specific postnatal growth promoting effects by IGF-II in the presence of elevated IGF-I     

Secretion of unprocessed human surfactant protein B in milk of transgenic mice

Because of the apparent clinical importance of human pulmonary surfactant B (SP-B), the expression of SP-B was directed to the mammary gland of transgenic mice using previously characterized rat whey acidic protein (WAP) regulatory sequences. rWAP/SP-B mRNA was expressed specifically in the mammary gland, and ranged from 1 to 5% of the endogenous WAP mRNA levels. SP-B was detected immunologically in both tissue and milk. The transgene product had an apparent molecular weight of 40--45 kDa, corresponding to the predicted size of the SP-B proprotein. Incubation of an SP-B-enriched fraction of milk with cathepsin D in vitro produced 20--25 kDa species, consistent with cleavage of the amino terminal domain by cathepsin D. This was confirmed using antibodies specific to the carboxy-terminal domain of SP-B. However, the appearance of only the SP-B proprotein in milk suggests that cathepsin D is not involved in the in vivo processing of SP-B. The SP-B proprotein can be expressed in milk of transgenic mice without any observed effects on mammary gland morphology or lactation