Sook-Jae Seo

Molecular characterization of the VLDL receptor homolog mediating binding of lipophorin in oocyte of the mosquito Aedes aegypti

Lipophorin (Lp) functions as a yolk protein precursor in the mosquito Aedes aegypti and it is internalized via receptor-mediated endocytosis (Insect Biochem. Mol. Biol., 30 (2000) 1161). We cloned and molecularly characterized a putative mosquito ovarian lipophorin receptor (AaLpRov) cDNA. The cDNA has a length of 3468 bp coding for a 1156-residue protein with a predicted molecular mass of 128.9 kDa. The deduced amino acid sequence of the cDNA revealed that it encodes a protein homolog of the LDL receptor superfamily, and that it harbors eight cysteine-rich ligand binding repeats at the N-terminus like vertebrate VLDL receptors. The deduced amino acid sequence of this mosquito ovarian receptor is most similar to that of the locust lipophorin receptor (LmLpR) (64.3%), and is only distantly related to the mosquito vitellogenin receptor (VgR) (18.3%), another ovarian LDLR homolog with a different ligand. The AaLpRov cDNA was expressed in a TnT Coupled Reticulocyte Lysate system, and co-immunoprecipitation experiments confirmed that the receptor protein specifically binds Lp. Developmental expression profiles clearly showed that AaLpRov transcripts are present in the vitellogenic ovary, with peak expression at 24-36 h post blood meal. In situ hybridization indicated that AaLpRov transcripts are present only in female germ line cells. Distance-based phylogenetic analyses suggest that the insect LpR and vertebrate LDL/VLDL receptor lineages separated after divergence from the insect VgR lineage.

cDNA sequence and gene expression of storage protein-2 — a juvenile hormone-suppressible hexamerin from the fall webworm, Hyphantria cunea Drury

We isolated and sequenced a cDNA clone corresponding to storage protein-2 (SP-2) from the fall webworm, Hyphantria cunea. The cDNA for SP-2 (2572 bp) codes for a 747-residue protein with a predicted molecular mass of 88.5 kDa. The calculated isoelectric point is 7.6. Multiple alignment analysis of amino acid sequence revealed that SP-2 is most similar to BJHSP2 (74.3% identity). According to both the phylogenetic analyses and criteria for amino acid composition, SP-2 belongs to the subfamily of moderately methionine-rich storage proteins (3.2% methionine, 11.8% aromatic amino acid). Topical application of the JH analog, methoprene, after head ligation of larvae, suppressed transcription of the SP-2 gene, indicating hormonal effects at the transcriptional level. The SP-2 transcript was detected by Northern blot analysis in Malpighian tubules, in addition to the fat body where it was most abundant. The local expression of SP-2 in Malpighian tubules suggests that it may have some function in that organ.

Incidence and polymerase chain reaction assay of Listeria monocytogenes from raw milk in Gyeongnam Province of Korea.

A total of 50 raw milk samples from Gyeongnam Province of Korea were examined for the incidence of Listeria monocytogenes between July 1998 and August 1998. L. monocytogenes isolated by biochemical test was confirmed by polymerase chain reaction (PCR) with two sets of primers designed from the invasion-associated protein (iap) gene. After standard PCR with external primers, the amplified DNA was confirmed by a second round of PCR with internal primers (nested PCR). Both the external and internal primers generated 468-bp and 287-bp products. respectively. Only one (G9 strain) of the three suspect samples that tested positive in biochemical tests for L. monocytogenes from 50 raw milk samples was also PCR positive. Following this procedure. PCR-positive G9 strain was confirmed by Southern blot using the 287-bp internal iap probe again. The detection limit of G9 strain by standard PCR assay was as few as 102 cells, equivalent to approximately I pg of L. monocytogenes DNA. These PCR assays may be useful for novel detection as well as rapid confirmation for L. monocytogenes from food samples and the field.

Fat body expressed yolk protein genes in Hyphantria cunea are related to the YP4 follicular epithelium yolk protein subunit gene of pyralid moths

cDNA clones for two of the yolk proteins, YP1 and YP2, produced by the fat body of the moth, Hyphantria cunea, were sequenced and found to be homologous to the follicular epithelium yolk proteins of pyralid moths. Both cDNA clones coded for polypeptides of 290 residues and the deduced amino acid sequence identity between YP1 and YP2 was very high (79.0%). Analysis of the secondary structure of the predicted polypeptides suggests that YP1 and YP2 do not form heteromeric proteins because of differences in secondary structure due to the lack of alpha helices in YP1. Northern blot analysis showed that the transcripts for YP1 (1.2 kb) and YP2 (1.1 kb) were present primarily in the female fat body with only trace levels detectable in the ovary of the adult female. In a developmental study, the YP1 and YP2 transcripts were first detectable in 10‐day‐old pupae and increased into the adult stage. These results suggest that the YP1 and YP2 genes in H. cunea have been recruited to replace the vitellogenin gene as the primary source of yolk proteins. During this process they have acquired a modified pattern of expression that is different from homologous genes reported in pyralid moths. The assessment of the evolution of proteinaceous yolk in these moths should serve as an excellent model for the evolution of gene recruitment.