Common Gene Products Research Articles

Lithium alters expression of RNAs in a type-specific manner in differentiated human neuroblastoma neuronal cultures, including specific genes involved in Alzheimer\u2019s disease

Lithium (Li) is a medication long-used to treat bipolar disorder. It is currently under investigation for multiple nervous system disorders, including Alzheimer’s disease (AD). While perturbation of RNA levels by Li has been previously reported, its effects on the whole transcriptome has been given little attention. We, therefore, sought to determine comprehensive effects of Li treatment on RNA levels. We cultured and differentiated human neuroblastoma (SK-N-SH) cells to neuronal cells with all-trans retinoic acid (ATRA). We exposed cultures for one week to lithium chloride or distilled water, extracted total RNA, depleted ribosomal RNA and performed whole-transcriptome RT-sequencing. We analyzed results by RNA length and type. We further analyzed expression and protein interaction networks between selected Li-altered protein-coding RNAs and common AD-associated gene products. Lithium changed expression of RNAs in both non-specific (inverse to sequence length) and specific (according to RNA type) fashions. The non-coding small nucleolar RNAs (snoRNAs) were subject to the greatest length-adjusted Li influence. When RNA length effects were taken into account, microRNAs as a group were significantly less likely to have had levels altered by Li treatment. Notably, several Li-influenced protein-coding RNAs were co-expressed or produced proteins that interacted with several common AD-associated genes and proteins. Lithium’s modification of RNA levels depends on both RNA length and type. Li activity on snoRNA levels may pertain to bipolar disorders while Li modification of protein coding RNAs may be relevant to AD.

A Burkholderia endophyte of the ancient maize landrace Chapalote utilizes c-di-GMP-dependent and independent signaling to suppress diverse plant fungal pathogen targets.

Chapalote is a maize (corn) landrace grown continuously by subsistence farmers in the Americas since 1000 BC, valued in part for its broad-spectrum pathogen resistance. Previously, we showed that Chapalote possesses a bacterial endophyte, Burkholderia gladioli strain 3A12, which suppresses growth of Sclerotinia homoeocarpa, a fungal pathogen of a maize relative, used as a model system. Ten mutants that lost the anti-pathogen activities were identified, corresponding to five genes. However, S. homoeocarpa is not a known maize pathogen; hence, the relevance of these anti-fungal mechanisms to its ancient host has not been clear. Here, the strain 3A12 mutants were tested against a known pathogen of maize and many crops, Rhizoctonia solani. Microscopy established that wild-type 3A12 swarms towards, and attaches onto, the pathogen, forming microcolonies, resulting in hyphal cleavage. Analysis of the mutants revealed that 3A12 uses common downstream gene products (e.g. fungicides) to suppress the growth of both S. homoeocarpa and R. solani, but apparently different upstream regulatory machinery, with the former, but not latter pathogen, requiring YajQ, a receptor for the secondary messenger c-di-GMP. We conclude that B. gladioli strain 3A12, an endophyte of an ancient maize, employs both c-di-GMP-dependent and independent signaling to target diverse fungal pathogens.

Spastic paraplegia proteins spastizin and spatacsin mediate autophagic lysosome reformation.

Autophagy allows cells to adapt to changes in their environment by coordinating the degradation and recycling of cellular components and organelles to maintain homeostasis. Lysosomes are organelles critical for terminating autophagy via their fusion with mature autophagosomes to generate autolysosomes that degrade autophagic materials; therefore, maintenance of the lysosomal population is essential for autophagy-dependent cellular clearance. Here, we have demonstrated that the two most common autosomal recessive hereditary spastic paraplegia gene products, the SPG15 protein spastizin and the SPG11 protein spatacsin, are pivotal for autophagic lysosome reformation (ALR), a pathway that generates new lysosomes. Lysosomal targeting of spastizin required an intact FYVE domain, which binds phosphatidylinositol 3-phosphate. Loss of spastizin or spatacsin resulted in depletion of free lysosomes, which are competent to fuse with autophagosomes, and an accumulation of autolysosomes, reflecting a failure in ALR. Moreover, spastizin and spatacsin were essential components for the initiation of lysosomal tubulation. Together, these results link dysfunction of the autophagy/lysosomal biogenesis machinery to neurodegeneration.

Dothistromin toxin is a virulence factor in dothistroma needle blight of pines

Dothistromin is a broad‐spectrum mycotoxin produced by the Dothideomycete pine needle pathogen Dothistroma septosporum. It accumulates in lesions, causing characteristic red bands on needles infected with this fungus. Dothistromin is similar in structure to the aflatoxin precursor versicolorin B and the biosynthetic pathways of these toxins share many common gene products. Although dothistromin is not essential for pathogenicity in dothistroma needle blight, its presence in infected needles suggests it might have a role in the disease process. The hypothesis that dothistromin is a virulence factor was tested by studying Pinus radiata infected with dothistromin‐deficient mutants of D. septosporum. The mutants were able to infect pine needles and complete their life cycle as previously shown, and were unaffected in spore germination, epiphytic growth or needle penetration. However, colonization of the mesophyll by the mutants was restricted compared to the wild type. Correspondingly, lesions produced by the mutants were smaller and produced significantly fewer spores than lesions produced by wildtype strains. Interestingly, ‘green islands’, in which chlorophyll was maintained at a higher level than in adjacent chlorotic and necrotic regions, surrounded early‐appearing lesions caused by both wildtype and mutant strains. At a later stage of disease development green islands were still present in the mutant but appeared to be masked by the extended dothistromin‐containing lesions in the wild type. Overall the results support a role for dothistromin in virulence in dothistroma needle blight.

Systematic Annotation and Bioinformatics Analyses of Large-Scale Oryza sativa Proteome

Much has been now recognized on the rice (Oryza sativa L.) proteomics by using the powerful experimental tool two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). 2D-PAGE can be utilized for monitoring global changes of quantitative protein expression in specific tissues under various conditions. However, systematic annotations of the protein spots generated by 2D-PAGE are still limited for rice. In this study, a new approach for Oryza sativa proteome annotation based on the 2D-gel maps was developed. Based on the publicly available 2D-PAGE data of rice, 11,201 gel spots were annotated accounting for 87.2% of the total spots on the gel maps. Gel spot alignments were performed for the annotated gel maps belonging to 23 rice tissues or organelles. In summary, 253 alignments between 23 tissues or organelles were performed, and 26,207 co-expressed proteins were identified using our analytical strategy. Large-scale bi-cluster analysis of 23 tissues/organelles proteomes of rice was carried out to detect novel functional proteins. Function and pathway analysis identified a number of common gene products with great potential in regulating specific physiological and biochemical events within various rice tissues/organelles. It also suggested that the tissue- or organelle-specific proteins might be responsible for the functional divergence of these tissues or organelles. Taken together, this study provides us new strategies and informative resources for rice proteome research based on 2D-PAGE data.

ClpP of Streptococcus mutans Differentially Regulates Expression of Genomic Islands, Mutacin Production, and Antibiotic Tolerance

Streptococcus mutans is the primary etiological agent of human dental caries and, at times, of infective endocarditis. Within the oral cavity, the pathogen is subjected to conditions of stress. A well-conserved protein complex named ClpP (caseinolytic protease) plays a vital role in adaptation under stress conditions. To gain a better understanding of the global role of the ClpP protease in cellular homeostasis, a transcriptome analysis was performed using a DeltaclpP mutant strain. The expression levels of more than 100 genes were up- or downregulated in the DeltaclpP mutant compared to the wild type. Notably, the expression of genes in several genomic islands, such as TnSmu1 and TnSmu2, was differentially modulated in the DeltaclpP mutant strain. ClpP deficiency also increased the expression of genes associated with a putative CRISPR locus. Furthermore, several stress-related genes and genes encoding bacteriocin-related peptides and many transcription factors were also found to be altered in the DeltaclpP mutant strain. A comparative analysis of the two-dimensional protein profile of the wild type and the DeltaclpP mutant strains showed altered protein profiles. Comparison of the transcriptome data with the proteomic data identified four common gene products, suggesting that the observed altered protein expression of these genes could be due to altered transcription. The results presented here indicate that ClpP-mediated proteolysis plays an important global role in the regulation of several important traits in this pathogen.

Rapid ??-1-antitrypsin M-variant Genotyping by Primer-induced Restriction Analysis

The 4 normal alleles of M1, M2, M3, and M4 are the most common gene products of the human alpha-1-antitrypsin (hAAT). Two single substitutions in M1 are responsible for M3 and M4, whereas 2 substitutions in M1 produce M2. Polymerase chain reaction-restriction fragment length polymorphism analysis of the Arg(101)/His(101) sequence variation can separate M1 and M3 from M2 and M4 alleles. To complete the genotyping procedure of hAAT M variants, the exon-V Glu(376)/Asp(376) sequence variation was directly analyzed using a designer primer with a single-base substitution in its sequence. This substitution induced an artificial site for the same restriction enzyme in the polymerase chain reaction product. The new restriction site was present in M1 and M4 but absent in M2 and M3, which can be applied as a rapid reliable means for the M-variant genotyping of hAAT.

Reporter gene transfer induces apoptosis in primary cortical neurons.

Modern cell biologists typically use reporter genes either alone or co-expressed with a protein of interest to facilitate the localization or quantification of protein expression. Our work demonstrates that reporter genes should be used cautiously, as several common reporter gene products are toxic to primary cortical neuronal cultures. We used the herpes simplex virus-based viral amplicon vector to transduce cortical neurons with three different reporter genes and assessed whether any reporter gene products were toxic over time, by monitoring neurite disintegration and apoptosis. Toxicity varied as a function of the reporter gene, the gene product localization, and the level of reporter gene expression. Transduction of enhanced green fluorescent protein or nuclear-localized beta-galactosidase was more toxic than non-nuclear localized beta-galactosidase. This work underscores the need for careful design of gene expression constructs. Moreover, in studies where cell injury or toxicity is being evaluated, their use should be carefully considered.

In vitro sulfotransferase activity of Rhizobium meliloti NodH protein: lipochitooligosaccharide nodulation signals are sulfated after synthesis of the core structure.

The Rhizobium common nod gene products NodABC are involved in the synthesis of the core lipochitooligosaccharide (Nod factor) structure, whereas the products of the host-specific nod genes are necessary for diverse structural modifications, which vary in different Rhizobium species. The sulfate group attached to the Rhizobium meliloti Nod signal is necessary for activity on the host plant alfalfa, while its absence renders the Nod factor active on the non-host plant vetch. This substituent is therefore a major determinant of host specificity. The exact biosynthetic pathway of Nod factors has not been fully elucidated. In particular, it is not known why some chemical modifications are introduced with high fidelity whereas others are inaccurate, giving rise to a family of different Nod factor structures produced by a single Rhizobium strain. Using protein extracts and partially purified recombinant NodH protein obtained from Escherichia coli expressing the R. meliloti nodH gene, we demonstrate here NodH-dependent in vitro sulfotransferase activity. Kinetic analyses with Nod factors, chitooligosaccharides, and their deacetylated derivatives revealed that Nod factors are the preferred substrate for the sulfate transfer. Moreover, the tetrameric Nod factor, NodRm-IV, was a better substrate than the trimer, NodRm-III, or the pentamer, NodRm-V. These data suggest that the core lipochitooligosaccharide structure must be synthesized prior to its host-specific modification with a sulfate group. Since in R. meliloti tetrameric Nod factors are the most abundant and the most active ones, high affinity of NodH for the appropriate tetrameric substrate guarantees its modification and thus contributes to the fidelity of host-specific behavior.

Parallel decrease of Na+ -taurocholate cotransport and its encoding mRNA in primary cultures of rat hepatocytes

We investigated the molecular mechanism underlying the progressive loss of Na(+)-dependent bile salt uptake in primary cultured rat hepatocytes. A specific cDNA probe was used to quantitate the levels of mRNA encoding the Na(+)-taurocholate-cotransporting polypeptide at various culture times. Hepatocytes were cultured on collagen in the presence of insulin (10(-7) mol/L), dexamethasone (10(-7) mol/L) and 10% fetal calf serum for up to 72 hr. During this time period the dissociation constant of Na(+)-dependent taurocholate uptake remained stable (19 to 39 mumol/L), whereas the maximum velocity values decreased from 100% at 3 hr to 55%, 22% and 4% at 24, 48 and 72 hr, respectively. Concomitantly the levels of the Na(+)-taurocholate-cotransporting polypeptide mRNA also decreased from 100% at 3 hr to 41%, 24% and 4% at the later time points. In contrast, Northern hybridization with complementary DNA probes for three common housekeeping gene products revealed a 1.8- to 3.4-fold increase in the levels of mRNA encoding the alpha-subunit of the Na+K(+)-ATPase, beta-actin and glycerol-3-phosphate dehydrogenase. These data indicate that the loss of Na(+)-dependent bile salt uptake in primary cultures of rat hepatocytes is caused by decreased levels of its specific mRNA. Hence the studies further confirm that without specific measures (primary) cultured rat hepatocytes reverse their liver-specific phenotype to a more fetal pattern of gene expression.

The biosynthesis of membrane and soluble plastidic c-type cytochromes of Chlamydomonas reinhardtii is dependent on multiple common gene products.

Cytochrome c6 functions in the thylakoid lumen to catalyze electron transfer from reduced cytochrome f of the cytochrome b6f complex to P700+ of photosystem I. The biogenesis of mature cyt c6 from cytosolically translated pre-apocytochrome c6 involves numerous post-translational modifications including the proteolytic removal of a transit sequence and the covalent attachment of heme to two cysteinyl thiols on the apoprotein. Here, we report on the characterization of a previously unrecognized class of non-allelic mutants of Chlamydomonas reinhardtii that are blocked at the conversion of apocyt c6 to holocyt c6. The mutants are acetate requiring since they are also deficient in cyt f, cyt b and the Rieske FeS protein. Pulse-chase studies indicate that heme attachment is not required for the two-step processing of pre-apocytochrome c6 to apocyt c6, but is required for the stability of the mature protein. This is in contrast to the biosynthesis of mitochondrial cyt c1 where heme attachment is required for the second processing step. We propose that the assembly of both holocytochrome c6 and the cytochrome b6f complex are dependent on common gene products, possibly those involved in heme delivery or metabolism. This is the first suggestion that multiple loci are involved in the biosynthesis of both plastidic c-type cytochromes.

Export of the outer membrane lipoprotein is defective in secD, secE, and secF mutants of Escherichia coli.

The export of major outer membrane lipoprotein has been found to be affected in secD, secE, and secF mutants of Escherichia coli, which are defective in protein export in general. After a shift to the nonpermissive temperature, the kinetics of accumulation of prolipoprotein and pre-OmpA protein was indistinguishable from that of pre-OmpA protein accumulation in the secD and secF mutants but different in the secE mutant. The prolipoprotein accumulated in the secD, secE, and secF mutants at the nonpermissive temperature was not modified with glyceride. We conclude from these results and those of previous studies that the export of lipoprotein requires all common sec gene products except the SecB protein, i.e., the SecA, SecD, SecE, SecF, and SecY proteins.

Use of avidin-biotin subtractive hybridization to characterize mRNA common to neurons destroyed by the selective neurotoxicant trimethyltin

Trimethyltin (TMT), a selective neurotoxicant, destroys a distinct subpopulation of neurons which possess no known biochemical or anatomic linkage. However, TMT-sensitive neurons may share common gene products related to susceptibility. In an effort to isolate mRNAs common to TMT-sensitive neurons, avidin/biotin based-subtractive hybridization was used to generate a cDNA library specifically related to TMT-toxicity. Out of 50 cDNAs, two clones hybridized only to poly(A +) mRNA isolated from the brains of saline-treated rats. Two of these cDNAs, p9T10 and p9T19, were used for in situ hybridization; both hybridized to hippocampus, limbic cortex, amygdala and other regions destroyed by TMT, suggesting that these probes identified mRNA enriched in TMT-sensitive neurons. The patterns of in situ hybridization coupled with the loss of p9T10 and p9T19 hybridization to mRNA isolated from the brains of TMT-treated rats suggests that one or both of these two clones may represent mRNA found in neurons damaged by TMT. The combination of selective neurotoxic lesions followed by cDNA subtractive hybridization should prove to be a useful strategy for the isolation of gene products from specific neuronal populations.

Electrophoretic polymorphism of human C4 is due to charge differences in the alpha chain, presumably in the C4d fragment.

Various common C4 gene products were isolated from serum by immunoprecipitation. After reduction the C4 alpha-, beta-, and gamma-polypeptide chains were studied by two-dimensional electrophoresis. Isoelectrofocusing was performed in the first dimension and sodium dodecyl sulphate polyacrylamide gradient gel electrophoresis in the second. The charge differences behind the electrophoretic C4 polymorphism were shown to reside in the 95,000-u (atomic mass units) alpha-chain. Charge variation closely mirroring the alpha-chain differences were also found in a 49,000-u fragment of the alpha-chain, most probably C4d. The basic beta-chain could not be studied in detail, but no differences were observed with regard to molecular weight or charge of the gamma-chains of the different C4 gene products.

Normal and mutant thermotaxis in the nematode Caenorhabditis elegans.

When grown at a temperature from 16 degrees to 25 degrees and placed on a thermal gradient, the nematode Caenorhabditis elegans migrates to its growth temperature and then moves isothermally. Behavioral adaptation to a new temperature takes several hours. Starved animals, in contrast, disperse from the growth temperature. Several mutants selected for chemotaxis defects have thermotaxis defects as well; these behaviors depend on some common gene products. New mutants selected directly for thermotaxis defects have unusual phenotypes which suggest mechanisms for thermotaxis.