In trypanosomes the homolog of the largest subunit of RNA polymerase II is encoded by two genes and has a highly unusual C-terminal domain structure

We have isolated the genes encoding the largest subunit of all three classes of RNA polymerase from Trypanosoma brucei. While the pol II largest subunit is encoded by a single gene in all organisms examined to date, trypanosomes contain two copies of the gene. Both genes are expressed in the procyclic and bloodstream stages of the trypanosome life cycle. The two pol II genes differ from one another in their coding sequences by 21 silent substitutions and 4 amino acid substitutions. In the core part of the large subunit, the predicted polypeptides are similar to other eukaryotic RNA polymerases. Both trypanosome pol II polypeptides, like those of other eukaryotes, also have a unique C-terminal extension. However, this domain in the trypanosome polypeptides, unlike those of other eukaryotes, is not a tandemly repeated heptapeptide sequence.     

Distribution and structure of cloned P elements from the Drosophila melanogaster P strain pi 2.

P transposable elements of Drosophila melanogaster cloned from the strong P strain pi 2 have been analysed. The structures and chromosomal locations of 26 of the 30-50 elements estimated to be present in pi 2 have been determined. At one location two elements are inserted 100 base pairs (bp) apart, and in a second location two elements are only separated by the 8 bp duplicated upon P-element insertion. In addition to 2.9 kilobase-pair (kbp) elements, elements with 14 different internal deletions from 1.3 to 2.3 kbp in size have been isolated. There are 7 copies of the 2.9 kbp element, 2 copies each of 5 internally deleted elements and a single copy of 9 internally deleted elements. One of the elements found twice is the KP element, which may play a role in the regulation of hybrid dysgenesis in strains which contain many copies of this element. Apart from internal deletions the elements are extremely homogeneous in DNA sequence, with only 2 single base polymorphisms detected twice each in over 16 kbp of P-element sequence. Although transpositions are infrequent in an inbred P cytotype strain such as pi 2, the distribution of these cloned elements indicates that when the genomic library was made, the strain was polymorphic with respect to element location. The distribution and structures of the element are discussed with respect to models for regulation of P-element transposition.     

A family of small repeated elements with some transposon-like properties in the genome of Neisseria gonorrhoeae

A physical technique known as two-dimensional S1 nuclease heteroduplex mapping has been applied to genomic DNA from the Gram-negative coccus Neisseria gonorrhoeae. This has resulted in the detection of two novel types of repetitive sequences. The first type is a repetitive sequence family of 152 base pairs (bp), whose ends are composed of inverted repeats of 26 bp. There are approximately 20 copies of this sequence, in both N. gonorrhoeae and Neisseria meningitidis (Correia, F., Inouye, S., and Inouye, M. (1986) J. Bacteriol. 167, 1009-1011). The second type of sequence is a 1443-bp duplication in the N. gonorrhoeae genome. The two classes of sequence are linked positionally. Each copy of the long duplicated sequence is adjacent to a member of the 152-bp repetitive sequence. In one instance two copies of the 152-bp repetitive sequence are separated by a 436-bp central region and are in an inverted orientation with respect to one another, resembling a compound transposable element.     

Pogo transposase contains a putative helix-turn-helix DNA binding domain that recognises a 12 bp sequence within the terminal inverted repeats.

Pogo is a transposable element with short terminal inverted repeats. It contains two open reading frames that are joined by splicing and code for the putative pogo transposase, the sequence of which indicates that it is related to the transposases of members of the Tc1/mariner family as well as proteins that have no known transposase activity including the centromere binding protein CENP-B. We have shown that the N-terminal region of pogo transposase binds in a sequence-specific manner to the ends of pogo and have identified residues essential for this. The results are consistent with a prediction that DNA binding is due to a helix-turn-helix motif within this region. The transposase recognises a 12 bp sequence, two copies of which are present at each end of pogo DNA. The outer two copies occur as inverted repeats 14 nucleotides from each end of the element, and contain a single base mismatch and indicate the inverted repeats of pogo are 26 nucleotides long. The inner copies occur as direct repeats, also with a single mismatch.     

Identification of the motifs within the tobacco mosaic virus 5''-leader responsible for enhancing translation.

The leader (called omega) of tobacco mosaic virus RNA enhances translation in both eukaryotes and prokaryotes. Although little secondary structure is predicted to exist within omega, the primary sequence of the 68 base leader is highly organized. Three copies of an eight base direct repeat and a (CAA)n region represent the two motifs found in the leaders of many TMV strains, and together these comprise 72% of omega. In previous deletion studies, no mutants exhibited loss-of-function, suggesting that functional redundancy exists within omega. We report here that a more comprehensive deletion analysis identified the motifs involved in translational enhancement. In a separate approach, oligonucleotides containing the sequence of each motif were used to construct leaders that varied in the number and configuration of the motifs. beta-Glucuronidase mRNA constructs containing these mutant leaders were synthesized in vitro and their translational efficiency measured in vivo following mRNA delivery to carrot protoplasts via electroporation. A combination of one copy of the 8 base direct repeat and a 25 base (CAA)n region was identified as the core regulatory element, although the (CAA)n motif is more critical. Two copies of the (CAA)n region are sufficient to confer a high level of enhancement and a leader composed of multiple copies of the direct repeat is moderately enhancing. Thus, these two motifs are functionally redundant.     

Origin and evolution of a major feline satellite DNA

A major satellite DNA has been cloned from the domestic cat (Felis catus) and characterized. The satellite monomer, termed FA-SAT, is 483 base-pairs in size, 64% G + C, and represents about 1 to 2% of the cat genome. A consensus sequence based upon partial sequence data from 21 independently isolated clones demonstrates: (1) FA-SAT is not composed of a series of shorter repeats, although about 25 copies, primarily imperfect, of the hexanucleotide TAACCC appear in the sequence; (2) there are many more CpG dinucleotides present in FA-SAT than expected for a random sequence of its size; and (3) 61% of all base substitutions in FA-SAT involve the replacement of G and C residues by A and T residues, indicating that FA-SAT is rapidly becoming A + T-rich. FA-SAT-related sequences are found in many mammals, where they appear to be scattered throughout the genome and not tandemly arranged as in the cat. An FA-SAT-related sequence was cloned from the domestic dog genome and sequenced, and shown to contain multiple copies of the same TAACCC hexanucleotide found in the cat satellite.     

IS186: an Escherichia coli insertion element isolated from a cDNA library.

Escherichia coli IS186 was isolated from cDNA libraries made from rainbow trout RNA and maintained in E. coli RR1. The element was 1,347 base pairs in length, had a perfect inverted repeat of 25 base pairs, and had an open reading frame of 375 amino acids. The hypothetical protein sequence of IS186 had limited homology to the E. coli IS4 hypothetical protein I sequence. There were three copies of IS186 in E. coli RR1.     

Protein assembly and DNA looping by the FokI restriction endonuclease

The FokI restriction endonuclease recognizes an asymmetric DNA sequence and cuts both strands at fixed positions upstream of the site. The sequence is contacted by a single monomer of the protein, but the monomer has only one catalytic centre and forms a dimer to cut both strands. FokI is also known to cleave DNA with two copies of its site more rapidly than DNA with one copy. To discover how FokI acts at a single site and how it acts at two sites, its reactions were examined on a series of plasmids with either one recognition site or with two sites separated by varied distances, sometimes in the presence of a DNA-binding defective mutant of FokI. These experiments showed that, to cleave DNA with one site, the monomer bound to that site associates via a weak protein–protein interaction with a second monomer that remains detached from the recognition sequence. Nevertheless, the second monomer catalyses phosphodiester bond hydrolysis at the same rate as the DNA-bound monomer. On DNA with two sites, two monomers of FokI interact strongly, as a result of being tethered to the same molecule of DNA, and sequester the intervening DNA in a loop.     

Sequence analysis of the insertion element ISH1.8 and of associated structural changes in the genome of phage PhiH of the archaebacterium Halobacterium halobium

We have sequenced the insertion element ISH1.8 which can be present in one or two copies in the genome of phage ΦH of Halobacterium halobium. ISH1.8 is 1895 bp long, has no inverted repeat at its ends, and one only of the two copies is flanked by two 5-bp duplications. An 8-bp sequence composed of 4 bp from each end of ISH1.8 is present in both sites lacking the element. This 8-bp sequence could either be a specific insertion sequence or a part of the element that is left behind upon deletion. The plasmid pΦHL, consisting of the invertible L segment of the phage genome which is, in ΦH2 and ΦH5, flanked by two copies of ISH1.8, contains 112 bp of ISH1.8 and is released from the phage genome by recombination within a direct repeat of 9 bp. This 9-bp sequence (TCCCGCCCT) exists as an inverted repeat in ISH1.8 and therefore as two distinct repeats in phage genomes containing two copies of ISH1.8 in inverted orientation.     

Tau, sigma, and delta. A family of repeated elements in yeast

We report here the isolation and structure of a new repeated DNA element, tau. This element, from Saccharomyces cerevisiae, is 371 base pairs long and is flanked on either end by the same invertedly repeated sequence found at the ends of some Ty and sigma elements in yeast, copia elements in Drosophila and spleen necrosis virus. The tau inverted repeats are themselves flanked by a 5-base pair directly repeated genomic sequence that is present only once in a cognate tau-allele. These structural characteristics, the presence of multiple copies of tau in the genome, and the isolation of tau+ and tau- allelic pairs suggest that tau may be capable of transposition either alone or in association with some larger element. Detailed sequence analysis of the tau, sigma, and delta elements revealed that all three contain significant regions of homology, suggesting that they are probably members of a single family derived from a common progenitor.     

The common 5'' terminal sequence on trypanosome mRNAs: a target for anti-messenger oligodeoxynucleotides.

Several mature mRNAs of Trypanosoma brucei were previously shown to have a common 5' terminal sequence of 35 nucleotides (nt) encoded by a separate mini-exon. To verify whether all trypanosome mRNAs contain this mini-exon sequence at their 5' end, we have tested oligodeoxynucleotides complementary to different parts of the 35 nt leader sequence for their ability to inhibit translation of total trypanosome mRNA. All oligomers tested inhibited translation of trypanosome mRNAs in a wheat germ extract. They had no effect on translation of Brome mosaic virus mRNA and of a trypanosome mRNA for phosphoglycerate kinase modified to remove the mini-exon sequence. Three different 12mers inhibited translation 35-60%; both the 22- and 34mer inhibited translation 95-100%. Incorporation of amino acids decreased proportionally in all protein bands detected in high resolution polyacrylamide gels. Our results show that all trypanosome mRNAs that yield a product detectable in gel contain a mini-exon sequence. We infer that most, if not all, trypanosome mRNAs contain a 5' terminal mini-exon sequence acquired by discontinuous synthesis.     

The 1723 element: a long, homogeneous, highly repeated DNA unit interspersed in the genome of Xenopus laevis

We describe a highly repeated DNA element in the Xenopus laevis genome. This sequence, named the 1723 element, was first identified among sequences that are transcribed during embryonic development. The element is present in about 8500 copies per haploid genome, which together accounts for about 2.4% of the genome. Most copies of the element have highly conserved restriction maps, and are interspersed in the genome. The copies range in size from 6000 to 10,000 base-pairs due to an expandable region that contains variable numbers of a tandemly repeating 183 to 204 base-pair unit. The element is framed by an imperfect 18 base-pair inverted sequence, and inverted repeats of 180 to 185 base-pairs are nearby. Sequence analysis of DNA adjacent to three cloned elements shows that the elements are flanked by 8 base-pair direct repeats. These and other properties of 1723 suggest that it may be transposable.     

Short repeated elements in the upstream regulatory region of the SUC2 gene of Saccharomyces cerevisiae.

Expression of secreted invertase from the SUC2 gene is regulated by carbon catabolite repression. Previously, an upstream regulatory region that is required for derepression of secreted invertase was identified and shown to confer glucose-repressible expression to the heterologous promoter of a LEU2-lacZ fusion. In this paper we show that tandem copies of a 32-base pair (bp) sequence from the upstream regulatory region activate expression of the same LEU2-lacZ fusion. The level of expression increased with the number of copies of the element, but was independent of their orientation; the expression from constructions containing four copies of the sequence was only twofold lower than that when the entire SUC2 upstream regulatory region was present. This activation was not significantly glucose repressible. The 32-bp sequence includes a 7-bp motif with the consensus sequence (A/C)(A/G)GAAAT that is repeated at five sites within the upstream regulatory region. Genetic evidence supporting the functional significance of this repeated motif was obtained by pseudoreversion of a SUC2 deletion mutant lacking part of the upstream region, including two copies of the 7-bp element. In three of five pseudorevertants, the mutations that restored high-level SUC2 expression altered one of the remaining copies of the 7-bp element.