Proteomic analysis of protein expression profiles during Caenorhabditis elegans development using two-dimensional difference gel electrophoresis

Coordinated protein expression is critical for the normal execution of animal development. To obtain overall proteome profiles during animal development, a small free-living soil nematode, Caenorhabditis elegans, was used as a model and the developmental changes of protein expressions were analyzed using two-dimensional difference gel electrophoresis. Protein samples from six developmental stages were prelabeled with fluorescent cyanine dyes and separated on two-dimensional electrophoresis gels. Image-to-image analysis of protein abundances together with protein identification by peptide mass fingerprinting yielded the developmental expression profiles of 231 spots representing 165 proteins. About a quarter of the identified proteins were expressed in multiple spots with different isoelectric points, suggesting a certain proportion of proteins were variously modified. This notion was supported by the observation that about a third of the multispot proteins were stained positive for a phosphoprotein specific dye. While a fairly large number of the proteins showed little alteration in their expression profiles during development, about 40 proteins were found to be significantly either up- or down-regulated between the embryos and newly hatched L1 larvae. Down-regulated proteins included those related to the cell cycle such as MCM-7, PCN-1, and the mitotic checkpoint protein, while up-regulated proteins included structural proteins such as actins, LEV-11, DIM-1, VAB-21, metabolic enzymes such as ATP synthase, ALH-12, fluctose-1,6-bisphosphate aldolase and GPD-3, and galectins. A standard proteome map was obtained where the defects in the mutations of developmental genes and the effects of reagents on the development in C. elegans were analyzed.     

Mouse U2af1-rs1 is a neomorphic imprinted gene.

The mouse U2af1-rs1 gene is an endogenous imprinted gene on the proximal region of chromosome 11. This gene is transcribed exclusively from the unmethylated paternal allele, while the methylated maternal allele is silent. An analysis of genome structure of this gene revealed that the whole gene is located in an intron of the Murr1 gene. Although none of the three human U2af1-related genes have been mapped to chromosome 2, the human homolog of Murr1 is assigned to chromosome 2. The mouse Murr1 gene is transcribed biallelically, and therefore it is not imprinted in neonatal mice. Allele-specific methylation is limited to a region around U2af1-rs1 in an intron of Murr1. These results suggest that in chromosomal homology and genomic imprinting, the U2af1-rs1 gene is distinct from the genome region surrounding it. We have proposed the neomorphic origin of the U2af1-rs1 gene by retrotransposition and the particular mechanism of genomic imprinting of ectopic genes.     

Genome mapping,molecular markers and marker-assisted selection in crop plants

Molecular Breeding -     

cDNA Sequences of Three Kinds of {beta}-tubulins from Rice

Complete nucleotide sequences of three kinds of rice ß-tubulincDNA clones (pTUB22, R1623 and R2242) were determined. Southernhybridization indicated that these ß-tubulins consistof one gene family. Using RFLP mapping, these three ß-tubulincDNAs were mapped to different chromosomes indicating at leastthree loci for the ß-tubulin gene. The deduced aminoacid sequences of these cDNAs showed a high similarity to otherplant ß-tubulins. The asparagine residue located atthe 100th amino acid from the Nterminus of plant ß-tubulinswas also conserved with these three ß-tubulins. Thisasparagine is thought to be responsible for the sensitivityagainst rhizoxin, the toxin of the pathogen of rice seedlingblight, Rhizopus sp. a soil-borne microorganism. Expressionof the three ß-tubulin genes was analyzed by Northernblotting and all three clones were expressed in root, the possibletarget tissue of rhizoxin. These results suggest that theseclones are candidates of ß-tubulins targeted by rhizoxin.     

Screening of RAPD Markers Linked to the Photoperiod-Sensitivity Gene in Rice Chromosome 6 Using Bulked Segregant Analysis

Bulked segregant analysis was used to determine randomly amplifiedpolymorphic DNA (RAPD) markers in a specific interval in themiddle of chromosome 6 of rice for tagging the photoperiod sensitivitygene.Two pools of F₂ individuals (japonica cv. Nipponbare and indicacv. Kasalath) were constructed according to the genotypes ofthree restriction fragment length polymorphism (RFLP) markerslocated at both ends and the middle of the targeted interval.Then another pair of pools were constructed based on the "graphicalgenotype," which was made with our high density linkage map.RAPD analysis was performed using these DNA pools as templates,and polymorphic fragments were detected and mapped. Using 80primers, either singlyor pairwise, we tested 2,404 primer pairsand established 14 markers tightly linked to the photoperiodsensitivitygene. The obtained RAPD markers were converted intosequence-tagged sites bycloning and sequencing of the polymorphicfragments and they can be used directlyfor construction of physicalmaps. This bulked segregant method can be applied for any speciesand any region of interest in which detailed linkage maps orphysical maps are needed.     

Specificity of trypsin digestion and conformational flexibility at different sites of unfolded lysozyme

Fourteen tryptic peptides and nine intermediates were identified as products of trypsin digestion of reduced and S-3-(trimethylated amino) propylated lysozyme. Kinetics of the appearance and disappearance of these products were observed by monitoring the peak areas on the chromatogram. In spite of the complicated reaction pathways, kinetics of the digestion of proteins and several intermediate products show simple decay curves with a single rate constant. In this paper, the trypsin susceptibility of the individual cleavage site is defined as a hydrolytic rate constant of the susceptible peptide bond in the presence of 10 nM trypsin. The cleavage sites of unfolded lysozyme are classified into two groups in terms of the trypsin susceptibility: one has a high susceptibility (10–20 h^?1) and the other a low susceptibility (1.0–2.0 h^?1). In the unfolded state of lysozyme, in conclusion, the region from residues 15 to 61 has a strong resistance to trypsin digestion; on the other hand, the C-terminal half of the polypeptide chain is flexible enough to fit into the active site of trypsin. In addition, six kinds of pentapeptides were synthesized as analogues of lysozyme fragments including Arg 14, Arg 21, Lys 33, Arg 45, Arg 61, and Arg 73. Kinetics of typtic digestion of them were observed. Both k_cat and K_M were determined for these synthetic pentapeptides. The susceptibility of each cleavage site in pentapeptides is determined and compared with that corresponding in proteins. The susceptibility is usually higher when the susceptible peptide chain is flexible. However, susceptibilities of a few sites in proteins are lower than those in pentapeptides. This means that the peptapeptides, this means that the peptide chains tend to fold locally to prevent trypsin from binding to the sites. It was found that the sites of Arg 21 and Arg 45 are indeed resistant to trypsin, but the site of Lys 33 is not so much, although the hydrolytic rate at Lys 33 itself is extremely slow. © 1994 John Wiley & Sons, Inc.     

Degradation of amyloid β peptide by neprilysin expressed from Borna disease virus vector

Accumulation of amyloid β (Aβ40 and Aβ42) in the brain is a characteristic of Alzheimer disease (AD). Because neprilysin (NEP) is a major Aβ‐degrading enzyme, NEP delivery in the brain is a promising gene therapy for AD. Borna disease virus (BoDV) vector enables long‐term transduction of foreign genes in the central nerve system. Here, the proteolytic ability of NEP transduced by the BoDV vector was evaluated and it was found that the amounts of Aβ40 and Aβ42 decreased significantly, suggesting that NEP expressed from the BoDV vector is functional in that it degrades Aβ.