Cloning and Expression Analysis of the pcbAB-pcbC β-Lactam Genes in the Marine Fungus Kallichroma tethys

ABSTRACT Here we report the identification of the β-lactam biosynthesis genes pcbAB and pcbC from a cosmid genomic DNA library of the marine fungus Kallichroma tethys. A BLAST homology search showed that they share high sequence identity with the δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetases and isopenicillin N synthases, respectively, of various fungal and bacterial β-lactam producers, while phylogenetic analysis indicated a close relationship with homologous genes of the cephalosporin-producing pyrenomycete Acremonium chrysogenum. Expression analysis by reverse transciption-PCR suggested that both genes are highly regulated and are expressed in the late growth phase of K. tethys cultures. Complementation of an Aspergillus nidulans strain deficient in ACV synthetase suggested that at least pcbAB is functional, although attempts to isolate active antibiotic from K. tethys were unsuccessful.

Felis domesticus papillomavirus, isolated from a skin lesion, is related to canine oral papillomavirus and contains a 1·3 kb non-coding region between the E2 and L2 open reading frames

We have characterized the complete genome (8300 bp) of an isolate of Felis domesticus papillomavirus (FdPV) from a domestic cat with cutaneous papillomatosis. A BLAST homology search using the nucleotide sequence of the L1 open reading frame demonstrated that the FdPV genome was most closely related to canine oral papillomavirus (COPV). A 384 bp non-coding region (NCR) was found between the end of L1 and the beginning of E6, and a 1·3 kbp NCR was located between the end of E2 and the beginning of L2. Phylogenetic analysis placed FdPV in the E3 clade with COPV. Both viruses contain the atypical second NCR, which has no homology with sequences in existing databases.

Differential expression of NAT1 translational repressor during development of bovine intramuscular adipocytes

This study was undertaken to test for differential gene expression in intramuscular adipocytes during fat deposition of feedlot steers. Angus × Hereford steers ( n = 50) were fed a high-energy concentrate ration ad libitum for 20 ( n = 5), 86 ( n = 15), 121 ( n = 15), and 146 days ( n = 15) to obtain various degrees of intramuscular adipocyte development. Carcass traits were significantly different ( P < 0.05) between the groups. Intramuscular adipose tissue was excised from the longissimus dorsi and snap frozen in liquid nitrogen. Pooled samples of total RNA representing each group were analyzed by differential-display polymerase chain reaction using 200 primer combinations comprising 20 arbitrary (5′) and 10 anchor (3′) oligonucleotides. Bands ( n = 70) representing putative differences among treatment groups were excised, sequenced, and subjected to BLAST homology search. From these, 40 contained significant homology to known genes. One was of particular interest, the translational repressor NAT1 (novel APOBEC-1 target-1). NAT1 mRNA was quantified in individual animals to confirm differential expression among treatment groups. Results indicate that NAT1 message is more abundant ( P < 0.05) in intramuscular adipocytes of younger/leaner animals.

Characterization of a novel genital human papillomavirus by overlapping PCR: candHPV86 identified in cervicovaginal cells of a woman with cervical neoplasia

A novel human papillomavirus (HPV), candHPV86, was cloned and characterized from cervicovaginal cells obtained from a 37-year-old Hispanic woman with cervical intraepithelial neoplasia grade 1 (CIN1) using an overlapping PCR technique. Primers were designed by phylogenetic alignment of closely related HPV genomes using the L1 fragment sequence amplified by GP5+/6+. The 7983 bp complete nucleotide sequence of the HPV genome was determined by sequence walking. A basic local alignment sequence tool (BLAST) homology search using the L1 open reading frame demonstrated that this HPV was most closely related to HPVHAN2294 (GenBank, AJ400628; 86% homology) and HPV84 (84% homology). candHPV86 was placed in the HPV genome homology group A3 by phylogenetic analyses. The overlapping PCR technique is applicable for characterizing the complete spectrum and variation of HPVs in a population.