Whole-genome organization and functional properties of miniature DNA insertion sequences conserved in pathogenic Neisseriae.

The chromosome of pathogenic Neisseriae is peppered by members of an abundant family of small DNA sequences known as Correia elements. These DNA repeats, that we call nemis (for neisseria miniature insertion sequences) can be sorted into two major size classes. Both unit-length (154-158 bp) and internally rearranged (104-108 bp) elements feature long terminal inverted repeats (TIRs), and can potentially fold into robust stem-loop structures. Nemis are (or have been) mobile DNA sequences which generate a specific 2-bp target site duplication upon insertion, and strictly recall RUP, a repeated DNA element found in Streptococcus pneumoniae. The subfamilies of 26L/26R, 26L/27R, 27L/27R and 27L/26R elements, found by wide-genome computer surveys in both the Neisseria meningitidis and the Neisseria gonorrhoeae genomes, originate from the combination of TIRs which vary in length (26-27 bp) as in sequence content (L and R types). In both species, the predominant subfamily is made by the 26L/26R elements. The number of nemis is comparable in the N. meningitidis Z2491 (A serogroup) and the MC58 (B serogroup) strains, but is sharply reduced in the N. gonorrhoeae strain F1090. Consequently, several genes which are conserved in the two pathogens are flanked by nemis DNA in the meningococcus genome only. More than 2/3 of nemis are interspersed with single-copy DNA, and are found at close distance from cellular genes. Both primer extension and RNase protection data lend support to the notion that nemis are cotranscribed with cellular genes and subsequently processed, at either one or both TIRs, by a specific endoribonuclease, which plausibly corresponds to RNase III.

The overlap of Inr and TATA elements sets the use of alternative transcriptional start sites in the mouse galectin-1 gene promoter.

In the mouse gene encoding the protein galectin-1, transcription initiation at the +1 site is directed by a TATA box. Here we show that a consensus Inr element (TCCAGTT), which spans residues -34 to -28 and overlaps the TATA box, directs RNA initiation also from a previously uncharacterized site located at position -31. Upstream transcripts are polyadenylated and contribute to more than half of the galectin-1 mRNA population in all tissues analyzed. The promoter architecture is evolutionarily conserved to man, and galectin-1 mRNA size variants accumulate also in human HeLa cells. The 5' end terminus of the transcripts initiated at residue -31 is extremely GC-rich, and may fold into a relative stable hairpin which could influence translation and thus modulate the intracellular levels of galectin-1. The interval -63/+45 contains sufficient information to ensure RNA initiation from both -31 and +1 sites, and a Sp1 site spanning residues -57 to -48 is crucial for promoter functioning. The unusual overlap of core promoter elements suggests that RNA initiation from the -31 and the +1 sites may take place in a sequential manner.

A variety of RNA polymerases II and III-dependent promoter classes is repressed by factors containing the Krüppel-associated/finger preceding box of zinc finger proteins.

KRAB/FPB (Krüppel-associated/finger preceding box) domains are small, portable transcriptional repression motifs, encoded by hundreds of vertebrates C2-H2-type zinc finger genes. We report that KRAB/FPB domains feature an unprecedented, highly promiscuous DNA-binding dependent transcriptional repressing activity. Indeed, template bound chimeric factors containing KRAB/FPB modules actively repress in vivo the transcription of distinct promoter classes that depend on different core elements, recruit distinct basal transcriptional apparatuses and are transcribed either by RNA polymerase II or III. The promoter types repressed in transient assays in a dose- and DNA-binding dependent, but position- and orientation-independent manner, by GAL4-KRAB/FPB fusions include an RNA polymerase II-dependent small nuclear RNA promoter (U1) as well as RNA polymerase III-dependent class 2 (adenovirus VA1), class 3 (human U6) and atypical (human 7SL) promoters. Down-modulation of all of these templates depended on factors containing the A module of the KRAB/FPB domain. Data provide further insights into the properties and mode of action of this widespread repression motif, and support the notion that genes belonging to distinct classes may be repressed in vivo by KRAB/FPB containing zinc finger proteins. The exquisitely DNA-binding dependent transcriptional promiscuity exhibited by KRAB/FPB domains may provide a unique model system for studying the mechanism by which a promoter recruited repression motif can down-modulate a large variety of promoter types.

Alternative activation of transcriptional initiators in Drosophila melanogaster LINE promoters.

In the Drosophila I, F and Doc LINEs, basal transcription is ensured by the functional interaction of initiator sequences with intragenic regulatory segments (B regions) which comprise distinct functional modules. Removing the B regions, as changing their composition or location, allowed different activators to stimulate transcription from novel initiators both in Doc and F promoters. The use of distinct initiators plausibly reflects the assembly of transcriptional complexes in which TFIID assumes alternative spatial conformations. The response of I, F and Doc promoters to the same enhancer is significantly influenced by the number, position and type of core elements present.

Multiple downstream promoter modules regulate the transcription of the Drosophila melanogaster I, Doc and F elements.

The basal promoters of three Drosophila long interspersed nuclear elements (LINEs), the I factor and the F and Doc elements, have the same architecture. In each, transcription is directed by an initiator which is faithfully and efficiently recognized only when flanked 3' by a DNA segment approximately 20 bp in length called the B region. The B regions of the three promoters are interchangeable and have a complex structure, comprising three functionally distinct elements: de1, de2 and de3. While de2 is relatively conserved, fitting the consensus RGACGTGY, de1 and de3 vary among the three promoters. At different levels, each downstream element is able to ensure accurate recognition of the initiator. The de2 domain stimulates transcription of the F, I and Doc promoters to the same extent. In contrast, the I de1 domain stimulates transcription much more efficiently than the corresponding domains of the F and Doc elements. The finding that de2 is selectively required in order to detect full activity of enhancer sequences found in the F element suggests that de1 and de2 interact with different proteins. The B regions can be replaced by and synergize with a TATA element, can functionally substitute for downstream promoter sequences in the Drosophila hsp70 gene, and significantly activate the mouse terminal deoxynucleotidyl transferase initiator. Our data suggest that the B regions stimulate transcription by providing sites of interaction for the TFIID complex. Sequences homologous to the del to de3 array are found downstream from the transcription start site(s) both in TATA-less and TATA-containing promoters.

Identification of sequences which regulate the expression of Drosophila melanogaster Doc elements.

Long interspersed nuclear elements (LINEs) are mobile DNA elements which propagate by reverse transcription of RNA intermediates. LINEs lack long terminal repeats, and their expression is controlled by promoters located inside to the transcribed region of unit-length DNA copies. Doc elements constitute one of the seven families of LINEs found in Drosophila melanogaster. Plasmids in which the chloramphenicol acetyltransferase (CAT) gene is preceded by DNA segments from different Doc family members were used as templates for transient expression assays in Drosophila S2 cells. Transcription is initiated at the 5' end of Doc elements within hexamers fitting the consensus (C/G)AYTCG and is regulated by a DNA region which is located approximately 20 base pairs (bp) downstream from the RNA start site(s). The region includes a sequence (RGACGTGY motif, or DE2) which stimulates transcription in other Drosophila LINEs, and two adjacent elements, DE1 and DE3. Moving the downstream region either 4 bp away from, or 5 bp closer to the RNA start site region inhibited transcription. Sequences located approximately 200 bp downstream from the Doc 5' end repressed CAT expression in an orientation- and position-dependent manner. The inhibition reflects impaired translation of the CAT gene possibly consequent to the interaction of specific Doc RNA sequences with a cellular component.

Expression of Drosophila melanogaster F elements in vivo.

Drosophila melanogaster F elements are mobile, oligo(A)-terminated DNA sequences that probably propagate by the retrotranscription of RNA intermediates. Polyadenylated transcripts corresponding in size to full-length (4.7 kb) family members were detected in the Drosophila melanogaster Canton-S strain from 2nd larval instar to the adult stage. RNA accumulation reached a maximum in pupae. In the adult, F elements are transcribed in both sexes. F expression is directed in vivo by the intragenic promoter (Fin) located at the 5' end of F. Whole-mount hybridizations were carried out to define the site of synthesis of full-length transcripts found in the ovary. Selective RNA accumulation was not detected in the cytoplasm of any specific cell type. Stained nuclear dots were observed in nurse cells from stage 2-3 to the end of oogenesis. RNase treatment of egg chambers prior to the addition of the probe led to disappearance of the nuclear dots and appearance of a cytoplasmic hybridization signal suggesting leakage of nuclear transcripts. Transgenic lines harbouring the chloramphenicol acetyltransferase (CAT) gene under the control of the Fin promoter were obtained. In independent lines, CAT enzyme levels mirror the ontogenetic profile of F expression drawn from Northern RNA blotting data. An antisense promoter (Fout) that is located downstream from the Fin promoter and transcribe too bords the 5' end of F seems to be constitutively expressed in the fly.

LINE-related elements in Drosophila melanogaster.

Mobile elements known as LINEs are members of a superfamily of repeated DNA conserved from protozoa to man. These sequences propagate by the retrotranscription of RNA intermediates and differ in many respects from retroviruses. Whereas most eukaryotic genomes host a single LINE family, several families of LINE-like sequences or type II retrotransposons coexist in the fruit fly Drosophila melanogaster. Properties and features of these elements are discussed in this work.

Functional dissection of two promoters that control sense and antisense transcription of Drosophila melanogaster F elements.

Drosophila melanogaster F elements are members of the super-family of LINEs, mobile repeated DNA sequences that lack LTRs and propagate by the reverse transcription of unit-length RNA intermediates. The F 5' end region harbours two promoters (F(in) and F(out)) that transcribe in a convergent manner. Each promoter has been functionally dissected by assaying in D. melanogaster cultured cells templates carrying base substitutions and/or deletions across the +1 to +245 region of the element F12. F(in), that likely controls the synthesis of gene products and transposition intermediates, is internal to the transcribed region. Two elements play a major role in F-sense transcription. The proximal element spans the interval +6 to +14 and includes a major RNA start site. Heterologous DNA featuring a nearly identical purine-pyrimidine sequence can functionally replace the initiator-like module only when properly spaced from downstream F sequences. The distal element is within the interval +18 to +46 and may correspond to a motif (AGACGTTT, +34 to +41) conserved in other Drosophila LINEs. F(out) is a TATA-less promoter that directs transcription predominantly from three nearby sites (a to c). F(out) expression is influenced by multiple elements located upstream of residue -68 relative to site a as +1 within a region (alpha) shown to stimulate the D. melanogaster hsp70 promoter in an orientation and position-independent fashion. Changes within the -43 to +24 interval may suppress or stimulate transcription from sites a and c. Initiation from a site approximately 30 nucleotides upstream of site a is enhanced by alterations of the interval -43 to -5. The expression of the two F promoters, determined by the interaction of the transcriptional machinery with distinct DNA sequences, is influenced by a common element within the alpha region.

Convergent transcription initiates from oppositely oriented promoters within the 5' end regions of Drosophila melanogaster F elements.

Drosophila melanogaster F elements are mobile, oligo(A)-terminated DNA sequences that likely propagate by the retrotranscription of RNA intermediates. Plasmids bearing DNA segments from the left-hand region of a full-length F element fused to the CAT gene were used as templates for transient expression assays in Drosophila Schneider II cultured cells. Protein and RNA analyses led to the identification of two promoters, Fin and Fout, that transcribe in opposite orientations. The Fin promoter drives the synthesis of transcripts that initiate around residue +6 and are directed toward the element. Fin, that probably controls the formation of F transposition RNA intermediates and gene products, is internal to the transcribed region. Sequences important for accumulation of Fin transcripts are included within the +1 to +30 interval; an additional regulatory element may coincide with a heptamer located downstream of this region also found in the 5' end regions of F-like Drosophila retrotransposons. Analysis of the template activity of 3' deletion derivatives indicates that the level of accumulation of Fin RNA is also dependent upon the presence of sequences located within the +175 to +218 interval. The Fout promoter drives transcription in the opposite orientation with respect to Fin. Fout transcripts initiate at nearby sites within the +92 to +102 interval. Sequences downstream of these multiple RNA start sites are not required for the activity of the Fout promoter. Deletions knocking out the Fin promoter do not impair Fout transcription; conversely, initiation at the Fin promoter still takes place in templates that lack the Fout promoter. At a low level, both promoters are active in cultured cells.

A cDNA clone homologous to Drosophila F elements from an imaginal discs poly(A)+ library.

A cDNA clone (F-id1) homologous to Drosophila F elements has been isolated from a cDNA library prepared from poly(A)+ RNA derived from imaginal discs. The F-id1 insert is identical to the 3' end terminus of Fw, an element inserted at the white locus upon irradiation. The isolation of F-id1 provides the first evidence that F elements are expressed in the fruit fly life.

Spacer promoters are orientation-dependent activators of pre-rRNA transcription in Drosophila melanogaster.

In Drosophila melanogaster, 240-base-pair (bp) repeats, clustered in tandem arrays within the ribosomal DNA nontranscribed spacer region, include sites of RNA polymerase I-dependent transcription initiation and elements that stimulate the rate of transcription from the downstream precursor rRNA (pre-rRNA) promoter. We have analyzed the in vivo transcriptional activity of a large set of recombinant constructs in which tandem arrays of distinct segments derived from a 240-bp repeat were inserted upstream of the pre-rRNA promoter. The results indicate that activating spacer elements are confined to a region of 70 bp. Enhancing units overlap with spacer promoters, since DNA segments that stimulate transcription at the gene promoter also efficiently drive transcription initiation. The finding that artificial spacer arrays invariably stimulate pre-rRNA transcription initiation in an orientation-dependent fashion suggest that spacer-initiated transcription is involved in the enhancement process. The minimal spacer activating segment includes a perfect copy of a core domain of the gene promoter extending from -24 to +10 flanked by poorly homologous upstream DNA sequences. Spacer and gene promoters are functionally interchangeable as activating units. However, the different combination of DNA elements within the two determines a functional hierarchy, as only the pre-rRNA promoter is responsive to the stimulatory action of upstream units.

Multiple repeated units in Drosophila melanogaster ribosomal DNA spacer stimulate rRNA precursor transcription.

Drosophila melanogaster ribosomal DNA (rDNA) transcriptional units are separated by nontranscribed spacer (NTS) segments consisting of tandemly arranged repeats 95, 330, and 240 base pairs long. NTS sequences stimulate transcription from the rRNA precursor (pre-rRNA) promoter. Primer extension analysis of RNA from cells cotransfected with plasmids carrying NTS sequences of various lengths shows that the activity of the pre-rRNA promoter is directly correlated with the number of 240-base-pair repeats; NTS sequences upstream of these units also stimulate pre-rRNA transcription. The NTS effect might depend upon transcription from duplicated promoters present within the 240- and 330-base-pair repeats. The strength of the pre-RNA promoter correlates in each construct with the level of spacer transcription. The action of spacer sequences, although able to take place over a large distance, is not independent of orientation: stimulation of pre-rRNA transcription is abolished in plasmids carrying inverted NTS segments. Removal of a putative transcription termination site located upstream of the pre-rRNA promoter has no effect on pre-rRNA initiation nor does it substantially alter spacer enhancement.

Close relationship between non-viral retroposons in Drosophila melanogaster.

G elements constitute one of the several moderately repeated DNA families of the Drosophila melanogaster genome. G elements lack terminal repetitions and structurally resemble mammalian processed pseudogenes because they terminate at one end in oligo-A tracts of variable length. G elements are mostly interspersed in the chromocentric heterochromatin with other repeated DNA sequences. Nucleotide sequence analysis of G3A, a family member inserted in a non-nucleolar rDNA unit, shows that functional G elements might have coding capacity for two polypeptides; one has homology to reverse transcriptases, the other is reminiscent of RNA binding proteins derived from the cleavage of retroviral gag polyproteins. Functionally related polypeptides are similarly encoded by members of two other Drosophila repeated DNA families, the F elements and the I factors. The similarity in structural organization and the relatedness of their potential gene products favors the hypothesis that G, F, and I sequences derive from a common ancestor and result from processes based on the reverse transcription of RNA intermediates that probably differ markedly from those ensuring the maintenance and dispersion of copia-like elements.

Related polypeptides are encoded by Drosophila F elements, I factors, and mammalian L1 sequences.

The structural organization of Drosophila F elements closely resembles that of L1 sequences, a major family of repetitive DNA elements dispersed in the genome of all mammals. Members of both families are flanked by target-site duplications of different length, vary in size because of heterogeneity at one end, and invariably terminate at the other end in characteristic adenosine-rich stretches often preceded by polyadenylylation signals. The nucleotide sequence of Fw, an F element found in the white locus of wi+A flies, reveals a large open reading frame upstream of the 3' adenosine-rich terminus encoding a possible reverse transcriptase homologous to those potentially encoded by functional L1 units and Drosophila I factors. A cysteine-rich region within an interrupted frame located at the 5' terminus of Fw suggests that complete F elements might additionally encode a nucleic acid binding protein. The observation that F elements and I factors encode functionally related polypeptides, and the extensive similarity of their hypothetical reverse transcriptases to L1 open reading frames, favors the hypothesis that all these sequences are evolutionarily related and transpose upon the cDNA conversion of RNA intermediates.

Transient expression of Drosophila melanogaster rDNA promoter into cultured Drosophila cells.

Recombinant plasmids that carry the bacterial CAT gene under the transcriptional control of the D. melanogaster rDNA promoter have been introduced by transfection into cultured Schneider II Drosophila cells and their template activity followed at the RNA and protein level. While no CAT enzyme activity is measurable 48 hrs after transfection, high levels of hybrid rRNA-CAT transcripts that originate at the authentic rRNA start site are detected by S1 mapping analysis. The interval -180/+34 of a rDNA transcriptional unit is sufficient to ensure faithful polymerase I transcription. However, the presence of a complete NTS (non transcribed spacer) region greatly enhances the transcriptional activity of exogenously added rDNA templates. Competition experiments between constructs carrying different amounts of NTS sequences indicate that spacer segments confer a transcriptional advantage efficiently attracting necessary transcription factors and/or polymerase I molecules.

Genomic and structural organization of Drosophila melanogaster G elements.

The properties and the genomic organization of G elements, a moderately repeated DNA family of D. melanogaster, are reported. G elements lack terminal repeats, generate target site duplications at the point of insertion and exhibit at one end a stretch of A residues of variable length. In a large number of recombinant clones analyzed G elements occur in tandem arrays, interspersed with specific ribosomal DNA (rDNA) segments. This arrangement results from the insertion of members of the G family within the nontranscribed spacer (NTS) of rDNA units. Similarity of the site of integration of G elements to that of ribosomal DNA insertions suggests that distinct DNA sequences might have been inserted into rDNA through a partly common pathway.

Transient expression of genes introduced into cultured cells of Drosophila.

Recombinant plasmids in which the sequence encoding the bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) has been placed under the control of Drosophila heat shock protein 70 (hsp 70) or copia promoters have been introduced into cultured cells of two Drosophila species (Schneider II line of Drosophila melanogaster and D. immigrans) as calcium-phosphate complexes. Within 1-2 days after transfection functional CAT enzyme was detected in cells exposed to either CAT recombinant. The expression of the bacterial information depends on the activity of the Drosophila promoters because plasmids in which the Drosophila DNA fragments were fused to the CAT coding sequence in inverted orientation did not support the synthesis of CAT enzyme activity. Low levels of CAT activity and of hybrid mRNA were detected in cells transformed with hsp-cat recombinants when the cells were maintained at room temperature, and both mRNA levels and CAT activity increased substantially after a brief exposure to 37 degrees C. hsp-cat mRNA has the same 5' terminus as authentic Drosophila hsp 70 messenger. These experiments document a practical system for the introduction and expression of isolated genes in cultured cells of Drosophila.

A family of oligo-adenylate-terminated transposable sequences in Drosophila melanogaster.

Five members of the F family of transposable elements in Drosophila melanogaster have been characterized. Together with earlier work, these experiments lead to the following conclusions. There are about 50 copies of F elements in the genome located at about 25 euchromatic sites and in the chromocenter. Three of the cloned F elements have a length of 4.7 X 10(3) bases, the other two are truncated at their left ends by 0.4 X 10(3) and 1.3 X 10(3) bases, the other two are truncated at their left ends by 0.4 X 10(3) and 1.3 X 10(3) bases. The shortest F element has been found in a recently arisen revertant of the white ivory mutation. All F elements terminate at their right ends in a stretch of 12 to 30 A residues. A polyadenylation signal (A-A-T-A-A-A) precedes this terminus. Analysis of homologous DNA regions derived from two different strains of D. melanogaster containing F elements (filled sites) or lacking F elements (empty sites) showed that F insertions lead to the generation of target site duplications of 8 to 13 base-pairs. The five target sites analyzed differ in sequence but show a low level of homology. F elements do not contain internal repeats and, in particular, they lack the terminal redundancy characteristic of many other transposable elements. We compare F elements with other oligo(A)-terminated sequences, specifically the Alu sequences and processed pseudogenes of mammals. The presence of an oligo(A) stretch and of a polyadenylation signal raises the possibility that F elements transpose by a mechanism that involves transcription and reverse transcription followed by reinsertion into the genome.