Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma.

https://www.jbc.org/content/279/50/52487.full.pdf

Mice Deficient in Sphingosine Kinase 1 Are Rendered Lymphopenic by FTY720

Functional conservation for lipid storage droplet association among Perilipin, ADRP, and TIP47 (PAT)-related proteins in mammals, Drosophila, and Dictyostelium.

We introduce a PCR-based procedure for generating a gene disruption construct. This method depends on DNA fragment fusion by the PCR technique and requires only two steps of PCR to obtain a sufficient amount of the gene disruption construct for one transformation experiment. The first step involves three separate PCR syntheses of a selectable marker cassette and the 5'- and 3'-regions of a target gene. Of the four primers used in amplification of the 5'- and 3'-regions of the target gene, two primers placed proximal to the site of the marker cassette are designed to have sequence tags complementary to the 5'- or 3'-side of the marker cassette. The two primers used in PCR synthesis of the marker cassette are complementary to the tagged primers. By fusion PCR, the 5' and 3' PCR products are linked to the marker cassette via the regions of tagged primers that overlap. A sufficient amount of the disruption construct can be directly amplified with the outermost primers. This method is simple, rapid and relatively inexpensive. In addition, there is the freedom of attaching long flanking regions to any selectable marker cassette.

PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors

Phg1p is a nine-transmembrane protein superfamily member involved in dictyostelium adhesion and phagocytosis.

In an effort to identify and characterize genes expressed during multicellular development in Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, li- brary S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other or- ganisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will pro- vide a useful resource for investigating the genetic networks that regulate multicellular development of this

Motonobu Yoshida

Papers

The Dictyostelium Developmental cDNA Project: Generation and Analysis of Expressed Sequence Tags from the First-Finger Stage of Development