Functional expression of a sequence-specific endonuclease encoded by the retrotransposon R2Bm

A fraction of the 28S ribosomal genes in certain insect species is interrupted by the insertion elements R1 and R2. These two elements from the silkworm Bombyx mori (R1Bm and R2Bm) are retrotransposons capable of transposing in a highly sequence-specific manner. We report here the functional expression in E. coli of the entire single open reading frame of R2Bm and show that it encodes a double-stranded endo-nuclease (integrase) that can specifically cleave the 28S gene at the R2 insertion site. The resulting cleavage is a 4 bp staggered 5' overhang. Deletion analysis of the 28S gene revealed that the DNA sequence required for specific cleavage is asymmetric with respect to the actual insertion (cleavage) site, with fewer than 10 bp required at one side and at least 24 bp at the other side of the site. A model is proposed based on these and previous data to account for the sequence-specific integration of the R2 retrotransposon.     

The primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus MHV-A59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism.

Sequence analysis of a substantial part of the polymerase gene of the murine coronavirus MHV-A59 revealed the 3' end of an open reading frame (ORF1a) overlapping with a large ORF (ORF1b; 2733 amino acids) which covers the 3' half of the polymerase gene. The expression of ORF1b occurs by a ribosomal frameshifting mechanism since the ORF1a/ORF1b overlapping nucleotide sequence is capable of inducing ribosomal frameshifting in vitro as well as in vivo. A stem-loop structure and a pseudoknot are predicted in the nucleotide sequence involved in ribosomal frameshifting. Comparison of the predicted amino acid sequence of MHV ORF1b with the amino acid sequence deduced from the corresponding gene of the avian coronavirus IBV demonstrated that in contrast to the other viral genes this ORF is extremely conserved. Detailed analysis of the predicted amino acid sequence revealed sequence elements which are conserved in many DNA and RNA polymerases.     

Bombyx mori 28S ribosomal genes contain insertion elements similar to the Type I and II elements of Drosophila melanogaster.

We have examined the 28S ribosomal genes of the silkmoth, Bombyx mori, for the presence of insertion sequences. Two types of insertion sequences were found, each approximately 5 kb in length, which do not share sequence homology. Comparison of the nucleotide sequences of the junction regions with the uninserted gene reveals that one type of insertion has resulted in a 14 bp duplication of the 28S coding region at the insertion site. The location of this insertion and the 14 bp duplication are identical to that found in the Type I ribosomal insertion element of Drosophila melanogaster. The second type of insertion element is located at a site corresponding to approximately 75 bp upstream of the first type. The location of this insertion, the variability detected at its 5' junction, and a short region of sequence homology at its 3' junction suggest that it is related to the Type II element of D. melanogaster. This is the first example of a Type II-like rDNA insertion outside of sibling species of D. melanogaster, and the first example of a Type I-like rDNA insertion outside of the higher Diptera.     

Molecular evolution of streptococcal M protein: cloning and nucleotide sequence of the type 24 M protein gene and relation to other genes of Streptococcus pyogenes.

The structural gene for the type 24 M protein of group A streptococci has been cloned and expressed in Escherichia coli. The complete nucleotide sequence of the gene and the 3' and 5' flanking regions was determined. The sequence includes an open reading frame of 1,617 base pairs encoding a pre-M24 protein of 539 amino acids and a predicted Mr of 58,738. The structural gene contains two distinct tandemly reiterated elements. The first repeated element consists of 5.3 units, and the second contains 2.7 units. Each element shows little variation of the basic 35-amino-acid unit. Comparison of the sequence of the M24 protein with the sequence of the M6 protein (S. K. Hollingshead, V. A. Fischetti, and J. R. Scott, J. Biol. Chem. 261:1677-1686, 1986) indicates that these molecules have are conserved except in the regions coding for the antigenic (type specific) determinant and they have three regions of homology within the structural genes: 38 of 42 amino acids within the amino terminal signal sequence, the second repeated element of the M24 protein is found in the M6 molecule at the same position in the protein, and the carboxy terminal 164 amino acids, including a membrane anchor sequence, are conserved in both proteins. In addition, the sequences flanking the two genes are strongly conserved.     

Site-specific ribosomal DNA insertion elements in Anopheles gambiae and A. arabiensis: nucleotide sequence of gene-element boundaries.

The nucleotide sequence of the junctions between the 28S ribosomal gene and site-specific insertion elements from two sibling mosquito species, Anopheles gambiae and A. arabiensis, is reported. In both species, elements insert at the same point within the 28S gene, but this site is 634 basepairs (bp) 3' of the R1 (Type I) insertion site in Drosophila melanogaster. The two mosquito elements each have poly A tails and a polyadenylation signal, but the extreme 3' and 5' ends show no other similarity to each other or to any other insertion element. In both mosquito species, identical target site duplications of 17 bp are generated. The sequence TNTCCCTNT found in this duplication is also found in the 14 bp target site duplications that flank R1 elements in D. melanogaster. Another sequence in this duplication, GGGATAACT, is very similar to the sequence GGGAGTAACT found in the 24 base sequence required by the Bombyx mori R2 endonuclease.     

The chitinase gene of the silkworm, Bombyx mori, contains a novel Tc-like transposable element

We have determined the cDNA sequence and the genomic organization of the chitinase gene of the silkworm, Bombyx mori. The cDNA encodes 544 amino acids having 83% amino acid homology to the chitinase of the tobacoo hornworm, Manduca sexta. The total length of the gene is larger than 25 kilobase pairs, and it is separated into 11 exons. The intron-exon boundaries are all in accordance with the GT-AG rule. Also, the TATA box sequence was found in the 5' upstream region of the gene, and the gene is mapped on the seventh chromosome. A novel DNA type transposon that shows similarity to the Tc-like element was found in the third intron in some strains of B. mori; other strains, however, lack this element in the same intron. This element has long terminal inverted repeats, presumably encodes a transposase of about 340 amino acids with a DDE motif, and has an amino-terminal domain with a strong nuclear localization function. Seven other transposable elements with homologous but distinct sequences were isolated from the B. mori genome. Together with plaque hybridization results, our findings suggest that these novel elements exist in multiple copies constituting a new Tc-like transposable element family in the silkworm genome.     

IS901, a new member of a widespread class of atypical insertion sequences, is associated with pathogenicity in Mycobacterium avium

An insertion sequence (IS901), found in pathogenic strains of Mycobacterium avium, but absent in M. avium complex isolates from patients with acquired immune deficiency syndrome (AIDS), has been isolated and sequenced. This insertion element has a nucleotide sequence of 1472 bp, with one open reading frame (ORF1), which codes for a protein of 401 amino acids. The amino acid sequence, terminal ends and target site of IS901 are similar to those of IS900, present in Mycobacterium paratuberculosis. However, the DNA sequences of these two IS elements exhibit only 60% homology, compared to a DNA homology of 98% between their respective hosts. IS901, like IS900, appears to belong to a family of related insertion elements present in actinomycetes and other bacteria. M. avium strains containing IS901 were found to be more virulent in mice than closely related strains lacking IS901. IS901 may be a useful tool for the study of the genetics of virulence in the M. avium complex and for obtaining stable integration of foreign genes into mycobacteria.