1.5 kb mRNA abundantly expressed in rat tumors encodes a 37 kilodalton protein in vitro

A cDNA clone, pAH34, corresponding to a 1.5 kb mRNA present abundantly in various rat tumors was examined for its protein coding capacity. Hybridization-selected RNAs from both poly(A)+ RNAs of a rat ascites hepatoma cell line, AH60C and of normal liver produced a polypeptide of 37 kilo daltons in vitro, but at much higher levels in the AH60C than in the normal rat liver. Two dimensional electrophoresis of the translation product revealed that the pI of this protein was 7.1. Nucleotide sequence analysis of pAH34 showed that the insert of the clone consisted of 462bp and contained the 3' portion of mRNA, including poly(A) stretch with AATAAA signal sequence centered 16 nucleotides upstream, a short untranslated region and an open reading frame corresponding to possibly 67 amino acids of the C-terminal portion.     

Interspersion of repetitive sequences in rat liver DNA

In rat liver DNA, which contains only 20% repetitive sequences, a close interspersion of repetitive and unique sequences is found in about 35 % of the total DNA. The mean length of repetitive and unique alternating sequences is respectively 230 and 400 base pairs.     

Analysis of rat repetitive DNA sequences.

Parameters of repetitive sequence organization have been measured in the rat genome. Experiments using melting, hydroxylapatite binding, and single strand specific nuclease digestion have been used to measure the number, length, and arrangement of repeated DNA sequences. Renaturation and melting or S1 nuclease digestion of 1.0 kbp DNA fragment show about 20% of rat DNA sequences are 3000-fold repeated. Renatured duplexes from 4.0 kbp DNA fragments display two repetitive size fractions after nuclease digestion. About 60% of the repeated sequences are 0.2-0.4 kbp long while the remainder are longer than 1.5 kbp. The arrangement of the repeated sequences has been measured by hydroxylapatite fractionation of DNA fragments of varying lengths bearing a repeated sequence. Repeated DNA sequences are interspersed among 2.5 kbp long nonrepeated sequences throughout more than 70% of the rat genome. There are approximately 350 different 3000-fold short repeated sequences in the rat interspersed among 600,000 nonrepeated DNA sequences.     

Microarray-based survey of repetitive genomic sequences in Vicia spp.

A modified DNA microarray-based technique was devised for preliminary screening of short fragment genomic DNA libraries from three Vicia species (V. melanops, V. narbonensis, and V. sativa) to isolate representative highly abundant DNA sequences that show different distribution patterns among related legume species. The microarrays were sequentially hybridized with labeled genomic DNAs of thirteen Vicia and seven other Fabaceae species and scored for hybridization signals of individual clones. The clones were then assigned to one of the following groups characterized by hybridization to: (1) all tested species, (2) most of the Vicia and Pisum species, (3) only a few Vicia species, and (4) preferentially a single Vicia species. Several clones from each group, 65 in total, were sequenced. All Group I clones were identified as rDNA genes or fragments of chloroplast genome, whereas the majority of Group II clones showed significant homologies to retroelement sequences. Clones in Groups III and IV contained novel dispersed repeats with copy numbers 10²–10⁶/1C and two genus-specific tandem repeats. One of these belongs to the VicTR-B repeat family, and the other clone (S12) contains an amplified portion of the rDNA intergenic spacer. In situ hybridization using V. sativa metaphase chromosomes revealed the presence of the S12 sequences not only within rDNA genes, but also at several additional loci. The newly identified repeats, as well as the retroelement-like sequences, were characterized with respect to their abundance within individual genomes. Correlations between the repeat distributions and the current taxonomic classification of these species are discussed.     

GroEL-GroES solubilizes abundantly expressed xylulokinase in Escherichia coli

AIMS: The aims of the present work were to solubilize the abundantly expressed recombinant xylulokinase in Escherichia coli and to develop a reliable xylulokinase assay. METHODS AND RESULTS: Three mutants of xylulokinase of Bacillus megaterium that were expressed at high level but formed insoluble protein in E. coli BL21(DE3)pLysS were selected for solubility study. The solubility of xylulokinase increased eight to 77-fold after introduction of molecular chaperones GroEL-GroES into the host. CONCLUSION: This investigation reports that GroEL-GroES minimizes the formation of insoluble protein in three highly expressed recombinant xylulokinases and an improved xylulokinase assay. SIGNIFICANCE AND IMPACT OF THE STUDY: Commercial production of bioethanol is critically dependent on the development of an efficient and low-cost process of enzymatic conversion of xylan, a major component in lignocellulose biomass, to xylulose-5-phosphate, which can then be channelled into pentose phosphate pathway and metabolized to ethanol. The improved intracellular xylulokinase activity is expected to facilitate the xylose degradation.     

Farnesyl diphosphate synthase is abundantly expressed and regulated by androgen in rat prostatic epithelial cells

Farnesyl diphosphate synthase (FPPS) has been identified as an androgen-response gene in the rat ventral prostate using a highly sensitive PCR-based cDNA subtraction technique. FPPS is an essential enzyme that catalyzes the synthesis of farnesyl diphosphate (FPP), which is required for cholesterol biosynthesis as well as protein prenylation. We have characterized the expression of FPPS in the rat prostate in response to androgen manipulation. Northern blot analysis showed that castration induced a 10-fold down-regulation of FPPS mRNA within 24 h in the ventral prostate and androgen replacement up-regulated FPPS mRNA rapidly in the regressed ventral prostate of a castrated rat. The expression of FPPS was also regulated by androgen in the lateral and dorsal prostate, indicating that FPPS is important to androgen action in all three lobes of the prostate. Western blot analysis showed that FPPS protein level was also regulated by androgen in the prostate. Northern blot analysis of tissue specificity indicated that FPPS was most abundantly expressed in the ventral prostate of a mature rat and was responsive to androgen manipulation in the prostate and seminal vesicles, but not in other tissues. In situ hybridization study showed that FPPS mRNA was localized to the prostatic epithelium. Interestingly, the expression of FPPS was elevated in Dunning rat prostate tumor cell lines. The above findings suggest that FPPS has the potential to play an important role in androgen action and prostate cancer progression.     

An unusual accumulation of repetitive sequences in the rat genome

We have found in the rat genomic DNA a fragment 1300 bp long, containing an unusual concentration of members of different repetitive families. Three different repeats were noticed. An Alu-like repeat, homologous to the mouse B1 sequence, was followed by a fragment containing alternating purine-pyrimidine bases as in Z-DNA. Finally, a third repeat was identified, containing 38 TAGA tetranucleotides as described for reptile DNA.     

Characterization of proteins expressed abundantly in the fruit-body ofFlammulina velutipes

Chronological changes of protein expression in the vegetative mycelium ofFlammulina velutipes and expression of these proteins in the fruit-body were investigated by two-dimensional polyacrylamide gel electrophoresis. Four proteins (FBA 1-4) expressed abundantly in the fruit-body were found to have different expression patterns in the vegetative mycelium after the fruiting treatment. FBA 1-4 had similar amino acid sequences and displayed a high similarity with the deduced amino acid sequence of theC1 cDNA, which has an Arg-Gly-Asp (RGD) cell-attachment sequence. This suggests that FBA 1-4 may have cell-to-cell attachment activity.     

Characterization of two abundantly expressed constitutive genes of Neurospora crassa

Two abundantly expressed, constitutive genes of Neurospora crassa were isolated during differential screening of Neurospora genomic libraries. The coding regions of these two genes, designated RLF1 and RLF3, were identified by hybridization of the cloned DNA sequences with cDNA probes made from polyadenylated RNA. The RLF3 gene was carried on a 15-kilobase Neurospora BamHI DNA fragment present in a lambda 1059 recombinant; a 2-kilobase restriction fragment that contains RLF3 was subcloned into plasmid pBR322 prior to further characterization. Southern blot analysis revealed that both RLF1 and RLF3 are single copy genes. Northern blot analysis and S1 nuclease mapping demonstrated that RLF1 is transcribed to yield a 1.6-kilobase RNA, whereas RLF3 appears to give rise to two distinct sized RNA species of 1.0 and 1.6 kilobases. RNA dot blot analysis provided conclusive evidence that both of these genes are constitutively expressed. These constitutive genes will be valuable to provide a detailed comparison with the 5' flanking regions of regulated genes.     

URB is abundantly expressed in adipose tissue and dysregulated in obesity

Dysregulated production of adipocytokines in obesity is involved in the development of metabolic syndrome. URB/DRO1 contains N-terminal signal sequence and is thought to play a role in apoptosis of tumor cells. In the present study, we investigated the expression pattern of URB mRNA in adipose tissue and secretion from cultured adipocytes. In human and mouse, URB mRNA was predominantly expressed in adipose tissue and was downregulated in obese mouse models, such as ob/ob, KKAy, and diet-induced obese mice. In 3T3L1 adipocytes, insulin, TNF-α, H₂O₂ and hypoxia decreased URB mRNA level. This regulation was similar to that for adiponectin and opposite to MCP-1. URB protein was secreted in media of URB cDNA-stably transfected cells and endogenous URB was detected in media of cultured human adipocytes. In conclusion, the expression pattern of URB suggests its role in obesity and the results suggest that URB is secreted, at least in part, from adipocytes.     

Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade

Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals.     

Methylation of repetitive DNA sequences in the brain during aging of the rat

Methylation of repetitive DNA sequences (RDS) of the genomic DNA of the brain of 15- and 88-week old rats was analysed by digestion with HpaII, MspI, EcoRI + HpaII and EcoRI + MspI followed by end-labelling. mCpG doublets are present in two RDS of approximately 5 and 0.4 kb, and are also randomly distributed throughout the genome. Hemimethylated mCpC doublets also occur. Both mCpG and mCpC doublets are found more in the old than in the young. This age-related increase in DNA methylation occurs both at CCGG sites of the RDS and in the entire genome. Such increase in DNA methylation may alter chromatin conformation and gene expression in the brain as the rat ages.     

Isolation of genes abundantly expressed in rice anthers at the microspore stage

A cDNA library of rice (Oryza sativa L.) has been constructed from anthers at an early stage of pollen development. By differential screening of the library, we have isolated cDNAs of two genes, designated as Osc4 and Osc6, that are abundantly expressed in anthers containing tetrads and uninucleate microspores, but are not expressed in leaves or roots. Expression of Osc4 is absent in mature anthers, while Osc6 is present although the expression decays during pollen maturation. A comparison of the nucleotide and deduced amino acid sequences with those in data banks has not shown significant homology to known molecules.     

A family of moderately repetitive sequences in mouse DNA.

When mouse DNA is digested to completion with restriction endonuclease Eco R1, a distinct band of 1.3 kb segments comprising about 0.5-3% of the genome is observed upon agarose gel electrophoresis. This DNA is not tandemly repeated in the genome and is not derived from mouse satellite DNA. Restriction endonuclease analysis suggested that the 1.3 kb segments are heterogeneous. Specific sequences were selected from the 1.3 kb segments and amplified by cloning in plasmid pBR322. Southern transfer experiments indicated that three separately cloned mouse DNA inserts hybridized predominantly to the Eco R1 1.3 kb band and to the conspicuous subsegments generated by secondary restriction endonuclease cleavage of the sucrose gradient purified 1.3 kb segments. Segments were also excised by Hha I (Hha I segments) from the chimeric plasmids containing mouse DNA inserts and subjected to restriction endonuclease and cross-hybridization analysis. It was found that the three Hha I segments were different, although two of them exhibited partial sequence homology. Cot analysis indicated that each of the Hha I segments are repeated about 10(4) times in the mouse genome. These findings indicate that a family of related but non-identical, moderately repetitive DNA sequences, rather than a single homogeneous repeat, is present in the 1.3 kb Eco R1 band.     

Chromosomal mapping of HSPCB and MYL1 expressed abundantly in the bovine fetus

Chromosomal mapping of expressed sequence tags for HSPCB and MYL1 expressed abundantly in the bovine fetus was performed by analyzing bovine/murine somatic cell hybrid DNAs with polymerase chain reaction (PCR) using primers specific for those 3'-untranslated regions. HSPCB and MYL1 were assigned to bovine chromosomes 23 and 2, respectively.