Cloning, nucleotide sequence, and expression of the HincII restriction-modification system.

Two genes, coding for the HincII from Haemophilus influenzae Rc restriction-modification system, were cloned and expressed in Escherichia coli RR1. Their DNA sequences were determined. The HincII methylase (M.HincII) gene was 1,506 base pairs (bp) long, corresponding to a protein of 502 amino acid residues (Mr = 55,330). The HincII endonuclease (R.HincII) gene was 774 bp long, corresponding to a protein of 258 amino acid residues (Mr = 28,490). The amino acid residues predicted from the R.HincII and the N-terminal amino acid sequence of the enzyme found by analysis were identical. These methylase and endonuclease genes overlapped by 1 bp on the H. influenzae Rc chromosomal DNA. The clone, named E. coli RR1-Hinc, overproduced R.HincII. The R.HincII activity of this clone was 1,000-fold that from H. influenzae Rc. The amino acid sequence of M.HincII was compared with the sequences of four other adenine-specific type II methylases. Important homology was found between tne M.HincII and these other methylases.     

Nucleotide sequence and genetic organization of the NgoPII restriction-modification system of Neisseria gonorrhoeae

Summary The NgoPII restriction endonuclease, which recognizes the sequence 5-GGCC-3, differs from its isoschizomer HaeIII in being sensitive to methylation at the external cytosine residue. The entire nucleotide sequence of a cloned 3.3 kb segment of Neisseria gonorrhoeae strain P9 chromosomal DNA which harbours the NgoPII restriction-modification system has been determined. This data, coupled with sub-cloning experiments, indicates that the restriction endonuclease (R.NgoII) and modification (M.NgoII) genes are transcribed from separate promoters but are arranged in tandem, with the R.NgoPII gene being located on the 5 side of the M.NgoPII gene. Unlike all previously reported restriction systems the 3 end of the endonuclease open reading frame overlaps the 5 end of the methylase open reading frame by 8 codons. This overlap may have implications for the regulation of the NgoPII restriction-modification system.     

The complete nucleotide sequence and its organization of the genome of Barley yellow dwarf virus-GAV

The complete nucleotide sequence of genomic RNA of BYDV-GAV was determined. It comprised 5685 nucleotides and contained six open reading frames and four un-translated regions. The size and organization of BYDV-GAV genome were similar to those of BYDV PAV-aus. The nucleotide and deduced amino acid sequences of the six ORFs were aligned and compared with those of other luteoviruses. The results showed that there was a high degree of identity between BYDV-GAV and MAV-PS1 in all ORFs except ORF5 and ORF6, which had only 87.4% and 70.2% identities respectively. The reported genomic nucleotide sequence of MAV was shorter than that of BYDV-GAV, but the comparison of the genomic nucleotide sequences for MAV-PS1 and GAV showed 90.4% sequence identity for the same region of the genome. According to the level of sequence similarities, BYDV-GAV should be closely related to BYDV-MAV.     

The complete nucleotide sequence and its organization of the genome of Barley yellow dwarf virus-GAV

The Barley yellow dwarf disease (BYD) was firstly recognized as an aphid transmitted virus disease by Oswald and Houston[1] in 1951. Now, Barley yel-low dwarf viruses (BYDVs) belong to members of the plant virus family Luteoviridae. They are phloem- limited and obligately transmitted in the circula-tive/persistent manner by several species of cereal aphids and can cause significant economic losses worldwide because of damage to barley, wheat, and oats. In China, BYDVs cause mainly yello…     

The complete nucleotide sequence and gene organization of carp (Cyprinus carpio) mitochondrial genome

The complete sequence of the carp mitochondrial genome of 16,575 base pairs has been determined. The carp mitochondrial genome encodes the same set of genes (13 proteins, 2 rRNAs, and 22 tRNAs) as do other vertebrate mitochondrial DNAs. Comparison of this teleostean mitochondrial genome with those of other vertebrates reveals a similar gene order and compact genomic organization. The codon usage of proteins of carp mitochondrial genome is similar to that of other vertebrates. The phylogenetic relationship for mitochondrial protein genes is more apparent than that for the mitochondrial tRNA and rRNA genes.Correspondence to: F. Huang     

The chloroplast genome of mulberry: complete nucleotide sequence, gene organization and comparative analysis

The complete nucleotide sequence of mulberry (Morus indica cv. K2) chloroplast genome (158,484 bp) has been determined using a combination of long PCR and shotgun-based approaches. This is the third angiosperm tree species whose plastome sequence has been completely deciphered. The circular double-stranded molecule comprises of two identical inverted repeats (25,678 bp each) separating a large and a small single-copy region of 87,386 bp and 19,742 bp, respectively. A total of 83 protein-coding genes including five genes duplicated in the inverted repeat regions, eight ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acids, were assigned on the basis of homology to predicted genes from other chloroplast genomes. The mulberry plastome lacks the genes infA, sprA, and rpl21 and contains two pseudogenes ycf15 and ycf68. Comparative analysis, based on sequence similarity, both at the gene and genome level, indicates Morus to be closer to Cucumis and Lotus, phylogenetically. However, at genome level, inclusion of non-coding regions brings it closer to Eucalyptus, followed by Cucumis. This may reflect differential selection pressure operating on the genic and intergenic regions of the chloroplast genome.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.Communicated by Y. Tsumura     

The XmnI restriction-modification system: Cloning,expression, sequence organization and similarity between the R and M genes

The xmnIRM genes from Xanthomonas manihotis 7AS1 have been cloned and expressed in Escherichia coli. The nucleotide (nt) sequences of both genes were determined. The XmnI methyltransferase (MTase)-encoding gene is 1861 by in length and codes for 620 amino acids (aa) (68660 Da). The restriction endonuclease (ENase)-encoding gene is 959 by long and therefore codes for a 319-aa protein (35275 Da). The two genes are aligned tail to tail and they overlap at their respective stop codons. About 4 × 10⁴ units/g wet cell paste of R·XmnI was obtained following IPTG induction in a suitable E. coli host. The xmnIR gene is expressed from the T7 promoter. M·XmnI probably modifies the first A in the sequence, GAA(N)₄TTC. The xmnIR and M genes contain regions of conserved similarity and probably evolved from a common ancestor. M·XmnI is loosely related to M·EcoRI. The XmnI R-M system and the type-I R-M systems probably derived from a common ancestor.     

The complete nucleotide sequence of the bacteriocinogenic plasmid CloDF13

The complete nucleotide sequence of the bacteriocinogenic plasmid CloDF13 has been determined. The plasmid consists of 9957 base pairs (molecular weight 6.64 X 10(6] with a GC content of 54.4%. At this moment 16 identified biological functions can be assigned to the primary structure of the CloDF13 DNA. The functions include those of eight protein encoding genes, two untranslated RNA species, and six DNA sites. We discuss these functions in relation to the structure of CloDF13 DNA. For convenience we have divided the CloDF13 genome into five defined regions: region I (origin of vegetative replication, priming and control of replication, type I incompatibility), region II (cloacin DF13, cloacin immunity, cloacin release, cloacin operon control), region III (double-stranded DNA-phage interaction, type II incompatibility, multimer resolution), region IV (inhibition of male specific RNA phages and transfer of Flac), and region V (mobility proteins, basis of mobility).     

First complete nucleotide sequence and heterologous gene organization of the two rRNA operons in the phytoplasma genome

Phytoplasmas are cell-wallless Gram-positive low G + C bacteria belonging to the Mollicutes that inhabit the cytoplasm of plants and insects. Although phytoplasmas possess two ribosomal RNA (rrn) operons, only one has been fully sequenced. Here, we determined the complete nucleotide sequence of both rrn operons (designated rrnA and rrnB) of onion yellows (OY) phytoplasma. Both operons have rRNA genes organized as 5'-16S-23S-5S-3' with very highly conserved sequences; the 16S, 23S, and 5S rRNA genes are 99.9, 99.8, and 99.1% identical between the two operons. However, the organization of tRNA genes in the upstream region from 16S rRNA gene and in the downstream region from 5S rRNA gene differs markedly. Several promoter candidates were detected upstream from both operons, which suggests that both operons are functional. Interestingly, both have a tRNA(Ile) gene in the 16S-23S spacer region, while the reported rrnB operon of loofah witches' broom phytoplasma does not, indicating heterogenous gene organization of rrnB within phytoplasmas. The phytoplasma tRNA gene organization is similar to that of acholeplasmas, a closely related mollicute, and different from that of mycoplasmas, another mollicute. Moreover, the organization suggests that the rrn operons were derived from that of a related nonmollicute bacterium, Bacillus subtilis. This data should shed light on the evolutionary relationships and phylogeny of the mollicutes.     

Cloning and complete nucleotide sequences of the type II restriction-modification genes of Salmonella infantis.

The complete type II restriction-modification system of Salmonella infantis was cloned in Escherichia coli as an R . Sau3AI fragment of 3,430 base pairs. The clone was shown to express the restriction endonuclease as well as the modification methylase. The nucleotide sequence of the above fragment showed two open reading frames of 461 and 230 codons in tail-to-tail orientation. These were shown to represent the modification methylase M . SinI and the restriction endonuclease R . SinI, respectively. The methylase M . SinI amino acid sequence revealed a considerable similarity to those of other deoxycytidylate methylases. In contrast, endonuclease R . SinI did not exhibit such a similarity to other restriction enzymes.