Cloning, sequencing and characterization of a chitin synthase gene fragment from the fungus Phascolomyces articulosus /

Phascolomyces articulosus genomic DNA was isolated from 48 h old hyphae and was used for amplification of a chitin synthase fragment by the polymerase chain reaction method. The primers used in the amplification corresponded to two widely conserved amino acid regions found in chitin synthases of many fimgi. Amphfication resulted in four bands (820, 900, 1000 and 1500 bp, approximately) as visualized in a 1.2% agarose gel. The lowest band (820 bp) was selected as a candidate for chitin synthase because most amplified regions from other fimgi so far exhibited similar sizes (600-750 bp). The selected fragment was extracted from the gel and cloned in the Hinc n site of pUC19. The derived plasmid and insert were designated ^\5C\9'PaCHS and PaCHS respectively. The plasmid pUC19-PaC/fS was digested by several restriction enzymes and was found to contain BamHl and HincU sites. Sequencing of PaCHS revealed two intron sequences and a total open reading frame of 200 amino acids. The derived polypeptide was compared with other related sequences from the EMBL database (Heidelberg, Germany) and was matched to 36 other fiilly or partially sequenced fimgal chitin synthase genes. The closest resemblance was with two genes (74.5% and 73.1% identity) from Rhizopus oligosporus. Southern hybridization with the cloned fragment as a probe to the PCR reaction showed a strong signal at the fragment selected for cloning and weaker signals at the other two fragments. Southern hybridization with partially digested Phascolomyces articulosus genomic DNA showed a single band. The amino acid sequence was compared with sequences from other chitin synthase gene classes using the CLUSTALW program. The chitin synthase fragment from Phascolomyces articulosus was initially grouped in class n along with chitin synthase fragments from Rhizopus oligosporus and Phycomyces blakesleeanus which also belong to the same class, Zygomycetes. Bootstrap analysis using the neighbor-joining method available by CLUSTALW verified such classification. Comparison of PaCHS revealed conservation of intron positions that are characteristic of chitin synthase gene fragments of zygomycetous fungi.

Heterogeneity of photosystem II as it occurs in domain specific regions of the thylakoid membrane of spinach (spinacia oleracia L.)

Thylakoid membrane fractions were prepared from specific regions of thylakoid membranes of spinach (Spinacia oleracea). These fractions, which include grana (83), stroma (T3), grana core (8S), margins (Ma) and purified stroma (Y100) were prepared using a non-detergent method including a mild sonication and aqueous two-phase partitioning. The significance of PSlla and PSII~ centres have been described extensively in the literature. Previous work has characterized two types of PSII centres which are proposed to exist in different regions of the thylakoid membrane. a-centres are suggested to aggregate in stacked regions of grana whereas ~-centres are located in unstacked regions of stroma lamellae. The goal of this study is to characterize photosystem II from the isolated membrane vesicles representing different regions of the higher plant thylakoid membrane. The low temperature absorption spectra have been deconvoluted via Gaussian decomposition to estimate the relative sub-components that contribute to each fractions signature absorption spectrum. The relative sizes of the functional PSII antenna and the fluorescence induction kinetics were measured and used to determine the relative contributions of PSlla and PSII~ to each fraction. Picosecond chlorophyll fluorescence decay kinetics were collected for each fraction to characterize and gain insight into excitation energy transfer and primary electron transport in PSlla and PSII~ centres. The results presented here clearly illustrate the widely held notions of PSII/PS·I and PSlIa/PSII~ spatial separation. This study suggests that chlorophyll fluorescence decay lifetimes of PSII~ centres are shorter than those of PSlIa centres and, at FM, the longer lived of the two PSII components renders a larger yield in PSlIa-rich fractions, but smaller in PSIlr3-rich fractions.

Locating and sequencing of the E3 and neighbouring regions in bovine advenovirus type 2

Adenoviruses are nonenveloped icosahedral shaped particles. The double stranded DNA viral genome is divided into 5 major early transcription units, designated E1 A, E1 B, and E2 to E4, which are expressed in a regulated manner soon after infection. The gene products of the early region 3 (E3), shown to be nonessential for viral replication in vitro, are believed to be involved in counteracting host immunosurveillance. In order to sequence the E3 region of Bovine adenovirus type 2 (BAV2) it was necessary to determine the restriction map for the plasmid pEA48. A physical restriction endonuclease map for BamHl, Clal, Eco RI, Hindlll, Kpnl, Pstt, Sail, and Xbal was constructed. The DNA insert in pEA48 was determined to be viral in origin using Southern hybridization. A human adenovirus type 5 recombinant plasmid, containing partial DNA fragments of the two transcription units L4 and L5 that lie just outside the E3, was used to localize this region. The recombinant plasmid pEA was subcloned to facilitate sequencing. The DNA sequences between 74.8 and 90.5 map units containing the E3, the hexon associated protein (pVIII), and the fibre gene were determined. Homology comparison revealed that the genes for the hexon associated pV11I and the fibre protein are conserved. The last 70 amino acids of the BAV2 pV11I were the most conserved, showing a similarity of 87 percent with Ad2 pV1I1. A comparison between the predicted amino acid sequences of BAV2 and Ad40, Ad41 , Ad2 and AdS, revealed that they have an identical secondary structure consisting of a tail, a shaft and a knob. The shaft is composed of 22, 15 amino acid motifs, with periodic glycines and hydrophobic residues. The E3 region was found to consist of about 2.3 Kbp and to encode four proteins that were greater than 60 amino acids. However, these four open reading frames did not show significant homology to any other known adenovirus DNA or protein sequence.