In vitro translation of maize ADH: Evidence for the anaerobic induction of mRNA

Total cellular RNA from anaerobically stressed maize seedling roots was used to stimulate in vitro translation of authentic maize alcohol dehydrogenase (ADH) in a rabbit reticulocyte lysate system. Total products from such reactions were displayed on NEPHGE-SDS two-dimensional gels and the Adhl-specific translation products were identified by using RNA from sib seedlings segregating for Adhl charge and size variants. The application of a rapid RNA isolation procedure allowed the efficient isolation of biologically active RNAs from small amounts of seedling material. Maize ADHs translated in vitro are identical in size to in vivo ADH. Further, no ADH was detected in the products of an in vitro translation reaction stimulated by total RNA from aerobically grown seedlings. This suggests that induction of ADH protein by anaerobic stress is accomplished by production of Adh mRNA rather than activation of sequestered mRNA. The mRNAs for maize ADH1 and ADH2 are among a small class of mRNAs induced during anaerobiosis.Research was supported by NSF Grant PCM 76-11009. M.D.B. is supported by National Institute of Health Grant PHS 5 T32 GM07227-04. R.J.F. is a Predoctoral Trainee in Genetics supported by National Institute of Health Training Grants 82 and 7757 from the National Institute of General Medical Sciences.     

Post-translational processing of 7S and 11S components of soybean storage proteins

The synthesis and processing of the major storage proteins in soybean cotyledons was studied both in vivo and in vitro. The and subunits of 7S as well as the 11S proteins are synthesized as higher molecular weight-precursors on membrane-bound polysomes. The initial translation products of the 7S are proteolytically cleaved during translation suggesting the removal of a signal peptide as evidenced by the presence of 2 and 2 peptides immunoreactive with 7S antibody in the in vitro chain completion products of the membrane-bound polysomes. This is followed or accompanied by cotranslational glycosylation, which increases their size equivalent to that of initially-synthesized precursors. In vivo pulse-labelled 7S and products are of slightly higher molecular weights than the immunoprecipitable chain-completion products, indicating further post-translational modifications. A slow post-translational processing during a period of 1.5 to 16 h yields the final 7S and glycoproteins.Acidic and basic subunits of the 11S protein appear to be synthesized from common large molecular weight (60K-59K) precursors. Antibodies to the 11S acidic component recognize both acidic and basic domains in the precursor while those raised against basic subunits appear to be specific for that region only. The processing of the 11S precursor is also very slow and occurs post-translationally. This slow rate of processing, coupled with a temporal difference in the synthesis of 7S and 11S components, suggests a highly coordinated mechanism for synthesis and packaging of these proteins into protein bodies during seed development.     

Genetics of lucilin,a storage protein from the sheep blowfly,Lucilia cuprina (Calliphoridae)

Lucilin, the main storage protein of larval fat body and hemolymph in the sheep blowfly, Lucilia cuprina, has been isolated as a series of trimers composed of subunits of 83,000±5% daltons. Extensive electrophoretically detectable polymorphism of lucilin subunit patterns occurs in wild and laboratory populations of Lucilia; from four to nine bands are seen in any one individual. Evidence from genetic, electrophoretic, immunological, and structural studies suggests the existence of a series of 12 or more closely related structural loci (designated Luc-1 to Luc-12) which may have arisen through gene duplication. Codominant allelic variation has been found at several of these loci. Luc-1 and Luc-3, and probably the other structural loci of the series, are located on chromosome 2.Financial support for this work was largely obtained through the Australian Research Grants Committee (Grant D65/15167). J. A. T. held an Honorary Fellowship at the Australian National University during 1972–1973.     

Inhibitory effect of naked neural BC1 RNA or BC200 RNA on eukaryotic in vitro translation systems is reversed by poly(A)-binding protein (PABP)

Regulated protein biosynthesis in dendrites of neurons might be a key mechanism underlying learning and memory. Neuronal dendritic BC1 RNA and BC200 RNA and similar small untranslated RNAs inhibit protein translation in vitro systems, such as rabbit reticulocyte lysate. Likewise, co-transfection of these RNAs with reporter mRNA suppressed translation levels in HeLa cells. The oligo(A)-rich region of all active small RNAs were identified as the RNA domains chiefly responsible for the inhibitory effects. Addition of recombinant human poly(A)-binding protein (PABP) significantly compensated the inhibitory effect of the small oligo(A)-rich RNA. In vivo, all BC1 RNA appears to be complexed with PABP. Nevertheless, in the micro-environment of dendritic spines of neuronal cells, BC1 RNPs or BC200 RNPs might mediate regulatory functions by differential interactions with locally limited PABP and/or directly or indirectly, with other translation initiation factors.     

Localization of mRNA for pea legumin:In situ hybridization using a biotinylated cDNA probe

Summary In situ hybridization has been used to locate mRNA, for the storage protein legumin, in cotyledon storage parenchyma tissue of developing pea (Pisum sativum L.) seeds. The mRNA was hybridized with a biotinylated probe of cDNA in pBR 322 and subsequently located by avidin conjugates. Avidin-rhodamine was used for fluorescence microscopy localization at a tissue/cellular level and avidin-peroxidase (with DAB) and avidin-ferritin compared for localization at an ultrastructural level. Specific fluorescence associated with avidin-rhodamine was distributed unevenly throughout the cytosol but the cell walls, starch grains, vacuoles and protein deposits were unstained. The sizes and distribution of the regions of higher labeling within the cytosol suggest an association with elements of the endomembrane system. Following DAB reaction of the specifically localized avidin-peroxidase most, although not all, stain product was associated with the endoplasmic reticulum. The ER-associated reaction product was also accumulated within the ER lumen.Avidin-ferritin was also localized both in the cytosol and in association with the endoplasmic reticulum, although was less readily visualized in cells with a conventional ultrastructural appearance.Localization of avidin-ferritin was more readily visualized in cells which had undergone some limited structural damage during specimen preparation. In such cases ferritin was also shown to be specifically associated with the transition vesicles and trans-face peripheral vesicles of some dictyosomes.     

Karyotype characterization of five Brazilian species of Echinodorus (Alismataceae) with chromosomal banding and 45S rDNA FISH

The karyotypes of five Brazilian species of Echinodorus; E. bolivianus, E. grandiflorus, E. longipetalus, E. macrophyllus and E. tenellus (Alismataceae); were studied using C-banding, CMA₃/DAPI banding and fluorescence in situ hybridization with a 45S rDNA probe. There were few differences in the G-C rich regions of the five species, but marked differences were seen in the number and position of C-bands, A-T rich regions and 45S rDNA sites. Overall, these characteristics were species-specific, with the exception of E. bolivianus and E. tenellus, which were highly similar in all of the karyotypic characteristics studied.     

Localization of liver fatty acid-binding protein and its mRNA in the liver and jejunum of rats: an immunohistochemical and in situ hybridization study

Summary The localization of liver fatty acid-binding protein (L-FABP) and its mRNA in the liver and jejunum was examined in normal and 3-day-fasted rats by means of immunohistochemistry using a specific antibody to L-FABP and in situ hybridization using a synthetic oligonucleotide complementary to L-FABP mRNA as probe. In the liver from normally fed rats, the signal for L-FABP mRNA in hepatocytes was distributed throughout the lobule, with higher intensity in the periportal than in the centrolobular region. After a 3-d fasting, the mRNA signal declined in intensity throughout the lobule, in accordance with the result of Northern blot analysis. Immunohistochemistry for L-FABP showed intralobular patterns of immunoreactivity similar to those of the mRNA signal in both fed and fasted animals. In the jejunum from fed rats, L-FABP-mRNA signal was abundant in the absorptive epithelial cells lining the lower two-thirds of villus and less abundant in the villus tip cells, while the intensity of L-FABP immunoreactivity remained high in the latter cells. Fasting brought about a downward shift of the mRNA signal to an area including the upper half of the crypt and the lower portions of villus, with decreased intensity in the rest of the villus. Immunohistochemistry also showed a downward extension of the immunoreactivity into the upper crypt area. The present results suggest that in situ hybridization is a useful tool to analyze regulations of the expression of L-FABP gene in the digestive organs in association with epithelial cell migration and dietary condition.     

Cells expressing preproenkephalin mRNA in the rat pineal gland are not serotonin-producing pinealocytes: Evidence usingin Situ hybridization combined with immunocytochemistry for serotonin

Summary 1. Preproenkephalin (PPEnk) mRNA expressing cells have been identified in rat pineal gland using radioactivein situ hybridization histochemistry. 2. Approximately 7% of the cells in the pineal gland (7.5±0.86, mean ± 95% CI) express PPEnk mRNA. These cells are distributed throughout the pineal as either scattered single cells or small groups of cells with large round or oval nuclei. 3. Usingin situ hybridization combined with ABC immunocytochemistry for serotonin (5-HT) in the same pineal sections, the PPEnk mRNA labeling cells are found not to be serotonin-immunoreactive cells. These data indicate that the PPEnk mRNA is expressed in a certain discrete subpopulation of cells in the rat pineal gland and these cells are not serotonin-producing pinealocytes. 4. The physiologic role of PPEnk-derived peptides in the pineal remains unknown. It is possible that these peptides either are synthesized and secreted as hormones or act as pineal paracrine signals.     

Changes in translatable poly(A) RNA from differentiated potato tissues transformed with shoot-inducing Ti TL-DNA of Agrobacterium tumefaciens

Summary Two dimensional gel electrophoresis was used to examine differences in steady state total poly(A) RNA from untransformed potato (Solanum tuberosum cv. Maris Bard) and potato transformed with shoot-inducing T_L-DNA from A. tumefaciens. RNA was compared from phenotypically very distinct in vitro cultured shoots, more similar grafted plants and tubers. In each case between 200–400 translation products were identified representing the more abundant poly(A) mRNA's. In general, poly(A) RNA from the transformed tissues gave more high molecular weight products. This increase was most evident in poly(A) RNA from shoot cultures. Depending on the tissue examined, 1–5% of the translation products with a molecular weight <43 KD were observed to increase or decrease in abundance. The influence of T-DNA on cellular gene expression in the different transformed potato tissues is discussed in relation to previously determined changes in T-DNA gene expression (particularly of the T-DNA cytokinin gene) and the corresponding changes in endogenous hormone concentrations. It is concluded that some of the specific changes in low molecular weight products are either directly caused by the increased cytokinin levels or are indirectly involved in maintaining the transformed phenotype. re]19850530 rv]19851206 ac]19851210     

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.     

Effects of pre-storage conditions on storage of in vitro cultures of Nephrolepis exaltata (L.) Schott and Cordyline fruticosa (L.) A. Chev.

In vitro cultures of Nephrolepis exaltata and Cordyline fruticosa were stored at 5°, 9° or 13°C, at a low irradiance (3–5 mol m^–2 s^–1) or in darkness. Prior to storage the cultures were subjected to 18°, 21°, 24° or 27°C and 15, 30 or 45 mol m^–2 s^–1 in a factorial combination.The optimal storage conditions for Nephrolepis were 9°C in complete darkness. These cultures were still transferable to a peat/perlite mixture at the end of the experimental period of 36 months.The optimal storage conditions for Cordyline were 13°C and a low light level (±3–5 mol m^-2 s^-1). When the pre-storage conditions were normal growth room conditions (24°C and 30 mol m^-2 s^-1), in vitro cultures could be stored for 18 months. With the most favourable pre-storage treatment (18°C and 15 mol m^-2 s^-1) some cultures still had green shoots after 36 months of storage, but did not survive transfer to peat/perlite.Pre-conditioning before storage was most favourable for Nephrolepis, and not that important, but still favourable, for Cordyline. There was an interaction between pre-storage temperature and pre-storage irradiance. For both species a high irradiance level was less favourable than a low irradiance level when combined with high growth room temperatures.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - NOA 2-naphthoxyacetic acid     

Translational control by the poly(A) binding protein: A check for mRNA integrity

The eukaryotic mRNA 3′ poly(A) tail and the 5′ cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the cap-binding protein eIF4E, and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this “closed loop” mRNA among other effects enhance the affinity of eIF4E for the 5′ cap, by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picornavirus’ internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to the formation of the initiation complex. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 4, pp. 684–693. The article is published in the original.     

Localization of substance P mRNA in cholinergic cells of the rat laterodorsal tegmental nucleus:In situ hybridization histochemistry and immunocytochemistry

1. In situ hybridization histochemical techniques in combination with immunocytochemistry and acetylcholinesterase (AChE) histochemistry were used to study the colocalization of messenger RNA (mRNA) encoding the neuropeptide substance P (SP) in cholinergic cells of the laterodorsal tegmental nucleus (LDT) of the rat pontine brain stem. 2. Alternate serial sections were hybridized with a 48-base, 35S-labeled synthetic oligonucleotide probe encoding SP using in situ hybridization histochemistry and processed either histochemically for AChE or immunocytochemically for choline acetyltransferase (ChAT). 3. In addition, serial section analysis was used to demonstrate the correlation between SP and SP mRNA in the same cells of the LDT. 4. These studies reveal that the cholinergic neurons of the LDT synthesize SP.     

High-resolution mapping of repetitive DNA by in situ hybridization: molecular and chromosomal features of prominent dispersed and discretely localized DNA families from the wild beet species Beta procumbens

Members of three prominent DNA families of Beta procumbens have been isolated as Sau3A repeats. Two families consisting of repeats of about 158 bp and 312 bp are organized as satellite DNAs (Sau3A satellites I and II), whereas the third family with a repeat length of 202 bp is interspersed throughout the genome. Multi-colour fluorescence in situ hybridization was used for physical mapping of the DNA families, and has shown that these tandemly organized families occur in large heterochromatic and DAPI positive blocks. The Sau3A satellite I hybridized exclusively around or near the centromeres of 10, 11 or 12 chromosomes. The Sau3A satellite family I showed high intraspecific variability and high-resolution physical mapping was performed on pachytene chromosomes using differentially labelled repeats. The physical order of satellite subfamily arrays along a chromosome was visualized and provided evidence that large arrays of plant satellite repeats are not contiguous and consist of distinct subfamily domains. Re-hybridization of a heterologous rRNA probe to mitotic metaphase chromosomes revealed that the 18S-5.8S-25S rRNA genes are located at subterminal position on one chromosome pair missing repeat clusters of the Sau3A satellite family I. It is known that arrays of Sau3A satellite I repeats are tightly linked to a nematode (Heterodera schachtii) resistance gene and our results show that the gene might be located close to the centromere. Large arrays of the Sau3A satellite II were found in centromeric regions of 16 chromosomes and, in addition, a considerable interspersion of repeats over all chromosomes was observed. The family of interspersed 202 bp repeats is uniformly distributed over all chromosomes and largely excluded from the rRNA gene cluster but shows local amplification in some regions. Southern hybridization has shown that all three families are specific for genomes of the section Procumbentes of the genus Beta.     

A nonradioactive whole-mount in situ hybridization protocol for Porphyra (Rhodophyta) gametophytic germlings

The objective of this study was to develop a method for whole-mount in situ hybridization (WISH) by using elongation factor-1 (EF-1) riboprobes in the red alga Porphyra yezoensis Ueda. Several modifications to the general WISH protocol, such as use of a short-length probe, performing partial digestion of the cell wall, optimization of proteinase K concentration and additional washing steps after hybridization were essential to reduce non-specific staining and to obtain sufficient quality of data. This protocol made it possible to detect a specific signal as a positive control in WISH assays of P. yezoensis.     

Molecular characterization of the 5S ribosomal gene of the <Emphasis Type="Italic">Bradysia hygida</Emphasis>(Diptera:Sciaridae)

The rRNA genes are amongst the most extensively studied eukaryotic genes. They contain both highly conserved and rapidly evolving regions. The aim of this work was to clone and to sequence the Bradysia hygida 5S rDNA gene. A positive clone was sequenced and its 346 bp sequence was analyzed against the GenBank database. Sequence analysis revealed that the B. hygida 5S (Bh5S) rDNA gene is 120 bp long and is 87% identical to the aphid Acyrthosiphon magnoliae 5S rDNA gene. The Bh5S rDNA gene presents two unusual features: a GG pair at the 5' end of the gene sequence and the localization of the polyT signal immediately after the 3' end of the gene. In situ5S hybridization experiments revealed that the Bh5S rDNA gene is localized in the autosomal A chromosome     

Restriction endonuclease digestion patterns of harvest mice (Reithrodontomys) chromosomes: a comparison to G-bands,C-bands,and in situ hybridization

Constitutive heterochromatin of a karyotypically conserved species of harvest mouse was compared to that of three karyotypically derived species of harvest mice by examining banding patterns produced on metaphase chromosomes with three restriction endonucleases (EcoRI, MboI and PstI). Banding patterns produced by two of these restriction endonucleases (EcoRI and MboI) were compared to published G- and C-banded karyotypes and in situ hybridization of a satellite DNA repeat for these taxa. The third restriction endonuclease (PstI) did not produce a detectable pattern of digestion. For the most part, patterns produced by EcoRI and MboI can be related to C-banded chromosomes and in situ hybridization of satellite DNA sequences. Moreover, digestion with EcoRI reveals bands not apparent with these other techniques, suggesting that restriction endonuclease digestion of metaphase chromosomes may provide additional insight into the structure and organization of metaphase chromosomes. The patterns produced by restriction endonuclease digestion are compatible with the chromosomal evolution of these taxa, documenting that in the highly derived taxa not only are the chromosomes rearranged but the abundance of certain sequences is highly variable. However, technical variation and difficulty in producing consistent results even on a single slide with some restriction endonucleases documents the problems associated with this method.