Accessory Sequences Research Articles

Plasmids carrying cloned fragments of RF DNA from the filamentous phage (phi)Lf can be integrated into the host chromosome via site-specific integration and homologous recombination.

Different regions of RF DNA from the filamentous bacteriophage phiLf were cloned in Escherichia coli vectors that can not be maintained in Xanthomonas. After introduction into X. campestris pv. campestris 17 (Xc17), most of these constructs were found to integrate into the host chromosome, either by recA-dependent homologous recombination or recA-independent site-specific integration. Mutations in himA, which codes for the alpha-subunit of the Integration Host Factor, does not affect the integration. Integration occurs into a chromosomal region which harbors a copy of a defective phage (4445 bp) that shares a high degree of identity with the phiLf genome. While various parts of the 4445-bp region are susceptible to homologous recombination, site-specific integration requires the attB sequence on the chromosome and the phage attP. The attB shows a high level of sequence identity (22 out of 28 bp) to the dif site required for E. coli Xer site-specific recombination, including the 6-bp central region, and 8/11 identity in both the left XerC-binding arm and the right XerD-binding arm, with the innermost 5 nt of the arms forming a dyad symmetry that is also present in dif. The attP has the same central region and shows 10/11 identity to the dif site in the left arm, but the sequence of the right arm is less conserved than that of attB. The smallest regions still capable of mediating integration are a cloned 72-bp phiLf attP-containing sequence and a 51-bp Xc17 attB-containing sequence, which was reinserted into the Xc17 chromosome after the 4445-bp region had been deleted, indicating that accessory sequences are not necessary and that the integrase required for site-specific integration is neither specified by the 4445-bp Xc17 chromosomal region nor encoded by the phiLf genome.

Two compound replication origins in Saccharomyces cerevisiae contain redundant origin recognition complex binding sites.

While many of the proteins involved in the initiation of DNA replication are conserved between yeasts and metazoans, the structure of the replication origins themselves has appeared to be different. As typified by ARS1, replication origins in Saccharomyces cerevisiae are <150 bp long and have a simple modular structure, consisting of a single binding site for the origin recognition complex, the replication initiator protein, and one or more accessory sequences. DNA replication initiates from a discrete site. While the important sequences are currently less well defined, metazoan origins appear to be different. These origins are large and appear to be composed of multiple, redundant elements, and replication initiates throughout zones as large as 55 kb. In this report, we characterize two S. cerevisiae replication origins, ARS101 and ARS310, which differ from the paradigm. These origins contain multiple, redundant binding sites for the origin recognition complex. Each binding site must be altered to abolish origin function, while the alteration of a single binding site is sufficient to inactivate ARS1. This redundant structure may be similar to that seen in metazoan origins.

A 5' splice site-proximal enhancer binds SF1 and activates exon bridging of a microexon.

Internal exon size in vertebrates occurs over a narrow size range. Experimentally, exons shorter than 50 nucleotides are poorly included in mRNA unless accompanied by strengthened splice sites or accessory sequences that act as splicing enhancers, suggesting steric interference between snRNPs and other splicing factors binding simultaneously to the 3' and 5' splice sites of microexons. Despite these problems, very small naturally occurring exons exist. Here we studied the factors and mechanism involved in recognizing a constitutively included six-nucleotide exon from the cardiac troponin T gene. Inclusion of this exon is dependent on an enhancer located downstream of the 5' splice site. This enhancer contains six copies of the simple sequence GGGGCUG. The enhancer activates heterologous microexons and will work when located either upstream or downstream of the target exon, suggesting an ability to bind factors that bridge splicing units. A single copy of this sequence is sufficient for in vivo exon inclusion and is the binding site for the known bridging mammalian splicing factor 1 (SF1). The enhancer and its bound SF1 act to increase recognition of the upstream exon during exon definition, such that competition of in vitro reactions with RNAs containing the GGGGCUG repeated sequence depress splicing of the upstream intron, assembly of the spliceosome on the 3' splice site of the exon, and cross-linking of SF1. These results suggest a model in which SF1 bridges the small exon during initial assembly, thereby effectively extending the domain of the exon.

X-ray structure of aminopeptidase A from Escherichia coli and a model for the nucleoprotein complex in Xer site-specific recombination.

The structure of aminopeptidase A (PepA), which functions as a DNA-binding protein in Xer site-specific recombination and in transcriptional control of the carAB operon in Escherichia coli, has been determined at 2.5 A resolution. In Xer recombination at cer, PepA and the arginine repressor (ArgR) serve as accessory proteins, ensuring that recombination is exclusively intramolecular. In contrast, PepA homologues from other species have no known DNA-binding activity and are not implicated in transcriptional regulation or control of site-specific recombination. PepA comprises two domains, which have similar folds to the two domains of bovine lens leucine aminopeptidase (LAP). However, the N-terminal domain of PepA, which probably plays a significant role in DNA binding, is rotated by 19 degrees compared with its position in LAP. PepA is a homohexamer of 32 symmetry. A groove that runs from one trimer face across the 2-fold molecular axis to the other trimer face is proposed to be the DNA-binding site. Molecular modelling supports a structure of the Xer complex in which PepA, ArgR and a second PepA molecule are sandwiched along their 3-fold molecular axes, and the accessory sequences of the two recombination sites wrap around the accessory proteins as a right-handed superhelix such that three negative supercoils are trapped.

Topology of Xer Recombination on Catenanes Produced by Lambda Integrase

Xer site-specific recombination at the psi site from plasmid pSC101 displays topological selectivity, such that recombination normally occurs only between directly repeated sites on the same circular DNA molecule. This intramolecular selectivity is important for the biological role of psi, and is imposed by accessory proteins PepA and ArcA acting at accessory DNA sequences adjacent to the core recombination site. Here we show that the selectivity for intramolecular recombination at psi can be bypassed in multiply interlinked catenanes. Xer site-specific recombination occurred relatively efficiently between antiparallel psi sites located on separate rings of right-handed torus catenanes containing six or more nodes. This recombination introduced one additional node into the catenanes. Antiparallel sites on four-noded right-handed catenanes, the normal product of Xer recombination at psi, were not recombined efficiently. Furthermore, parallel psi sites on right-handed torus catenanes were not substrates for Xer recombination. These findings support a model in which psi sites are plectonemically interwrapped, trapping a precise number of supercoils that are converted to four catenation nodes by Xer strand exchange.

Definition of the transcriptional activation domains of three human HOX proteins depends on the DNA-binding context.

Hox proteins control developmental patterns and cell differentiation in vertebrates by acting as positive or negative regulators of still unidentified downstream target genes. The homeodomain and other small accessory sequences encode the DNA-protein and protein-protein interaction functions which ultimately dictate target recognition and functional specificity in vivo. The effector domains responsible for either positive or negative interactions with the cell transcriptional machinery are unknown for most Hox proteins, largely due to a lack of physiological targets on which to carry out functional analysis. We report the identification of the transcriptional activation domains of three human Hox proteins, HOXB1, HOXB3, and HOXD9, which interact in vivo with the autoregulatory and cross-regulatory enhancers of the murine Hoxb-1 and human HOXD9 genes. Activation domains have been defined both in a homologous context, i.e., within a HOX protein binding as a monomer or as a HOX-PBX heterodimer to the specific target, and in a heterologous context, after translocation to the yeast Gal4 DNA-binding domain. Transfection analysis indicates that activation domains can be identified in different regions of the three HOX proteins depending on the context in which they interact with the DNA target. These results suggest that Hox proteins may be multifunctional transcriptional regulators, interacting with different cofactors and/or components of the transcriptional machinery depending on the structure of their target regulatory elements.

Diverse amino acid residues function within the type 1 peroxisomal targeting signal. Implications for the role of accessory residues upstream of the type 1 peroxisomal targeting signal.

The purpose of this study was to determine whether the plant type 1 peroxisomal targeting signal (PTS1) utilizes amino acid residues that do not strictly adhere to the serine-lysine-leucine (SKL) motif (small-basic-hydrophobic residues). Selected residues were appended to the C terminus of chloramphenicol acetyltransferase (CAT) and were tested for their ability to target CAT fusion proteins to glyoxysomes in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 suspension-cultured cells. CAT was redirected from the cytosol into glyoxysomes by a wide range of residues, i.e. A/C/G/S/T-H/K/ L/N/R-I/L/M/Y. Although L and N at the -2 position (-SLL, -ANL) do not conform to the SKL motif, both functioned, but in a temporally less-efficient manner. Other SKL divergent residues, however, did not target CAT to glyoxysomes, i.e. F or P at the -3 position (-FKL, -PKL), S or T at the -2 position (-SSI, STL), or D at the -1 position (-SKD). The targeting inefficiency of CAT-ANL could be ameliorated when K was included at the -4 position (-KANL). In summary, the plant PTS1 mostly conforms to the SKL motif. For those PTS1s that possess nonconforming residue(s), other residues upstream of the PTS1 appear to function as accessory sequences that enhance the temporal efficiency of peroxisomal targeting.

Direct interaction of aminopeptidase A with recombination site DNA in Xer site-specific recombination.

Xer site-specific recombination at ColE1 cer converts plasmid multimers into monomers, thus ensuring the heritable stability of ColE1. Two related recombinase proteins, XerC and XerD, catalyse the strand exchange reaction at a 30 bp recombination core site. In addition, two accessory proteins, PepA and ArgR, are required for recombination at cer. These two accessory proteins are thought to act at 180 bp of accessory sequences adjacent to the cer recombination core to ensure that recombination only occurs between directly repeated sites on the same molecule. Here, we demonstrate that PepA and ArgR interact directly with cer, forming a complex in which the accessory sequences of two cer sites are interwrapped approximately three times in a right-handed fashion. We present a model for this synaptic complex, and propose that strand exchange can only occur after the formation of this complex.

Pyrimidine tracts between the 5' splice site and branch point facilitate splicing and recognition of a small Drosophila intron.

The minimum size for splicing of a vertebrate intron is approximately 70 nucleotides. In Drosophila melanogaster, more than half of the introns are significantly below this minimum yet function well. Such short introns often lack the pyrimidine tract located between the branch point and 3' splice site common to metazoan introns. To investigate if small introns contain special sequences that facilitate their recognition, the sequences and factors required for the splicing of a 59-nucleotide intron from the D. melanogaster mle gene have been examined. This intron contains only a minimal region of interrupted pyrimidines downstream of the branch point. Instead, two longer, uninterrupted C-rich tracts are located between the 5' splice site and branch point. Both of these sequences are required for maximal in vivo and in vitro splicing. The upstream sequences are also required for maximal binding of factors to the 5' splice site, cross-linking of U2AF to precursor RNA, and assembly of the active spliceosome, suggesting that sequences upstream of the branch point influence events at both ends of the small mle intron. Thus, a very short intron lacking a classical pyrimidine tract between the branch point and 3' splice site requires accessory pyrimidine sequences in the short region between the 5' splice site and branch point.

DNA sequence of recombinase-binding sites can determine Xer site-specific recombination outcome.

Xer site-specific recombination functions in the stable inheritance of circular plasmids and bacterial chromosomes. Two related recombinases, XerC and XerD, mediate this recombination, which 'undoes' the potential damage of homologous recombination. Xer recombination on natural plasmid sites is preferentially intramolecular, converting plasmid multimers to monomers. In contrast, recombination at the Escherichia coli recombination site, dif, occurs both intermolecularly and intramolecularly, at least when dif is inserted into a multicopy plasmid. Here the DNA sequence features of a family of core recombination sites in which the XerC- and XerD-binding sites, which are separated by 6 bp, were analysed in order to ascertain what determines whether recombination will be preferentially intramolecular, or will occur both within and between molecules. Sequence changes in either the XerC- or XerD-binding site can alter the recombination outcome. Preferential intramolecular recombination between a pair of recombination sites requires additional accessory DNA sequences and accessory recombination proteins and is correlated with reduced affinities of recombinase binding to recombination core sites, reduced XerC-mediated cleavage in vitro, and an apparent increased overall bending in recombinase-core-site complexes.

Determinants of selectivity in Xer site-specific recombination.

A remarkable property of some DNA-binding proteins that can interact with and pair distant DNA segments is that they mediate their biological function only when their binding sites are arranged in a specific configuration. Xer site-specific recombination at natural plasmid recombination sites (e.g., cer in ColE1) is preferentially intramolecular, converting dimers to monomers. In contrast, Xer recombination at the Escherichia coli chromosomal site dif can occur intermolecularly and intramolecularly. Recombination at both types of site requires the cooperative interactions of two related recombinases, XerC and XerD, with a 30-bp recombination core site. The dif core site is sufficient for recombination when XerC and XerD are present, whereas recombination at plasmid sites requires approximately 200 bp of adjacent accessory sequences and accessory proteins. These accessory factors ensure that recombination is intramolecular. Here we use a model system to show that selectivity for intramolecular recombination, and the consequent requirement for accessory factors, can arise by increasing the spacing between XerC- and XerD-binding sites from 6 to 8 bp. This reduces the affinity of the recombinases for the core site and changes the geometry of the recombinase/DNA complex. These changes are correlated with altered interactions of the recombinases with the core site and a reduced efficiency of XerC-mediated cleavage. We propose that the accessory sequences and proteins compensate for these changes and provide a nucleoprotein structure of fixed geometry that can only form and function effectively on circular molecules containing directly repeated sites.

Site-specific recombination and circular chromosome segregation

The Xer site-specific recombination system functions in Escherichia coli to ensure that circular plasmids and chromosomes are in the monomeric state prior to segregation at cell division. Two recombinases, XerC and XerD, bind cooperatively to a recombination site present in the E. coli chromosome and to sites present in natural multicopy plasmids. In addition, recombination at the natural plasmid site cer, present in ColEl, requires the function of two additional accessory proteins, ArgR and PepA. These accessory proteins, along with accessory DNA sequences present in the recombination sites of plasmids are used to ensure that recombination is exclusively intramolecular, converting circular multimers to monomers. Wild-type and mutant recombination proteins have been used to analyse the formation of recombinational synapses and the catalysis of strand exchange in vitro. These experiments demonstrate how the same two recombination proteins can act with different outcomes, depending on the organization of DNA sites at which they act. Moreover, insight into the separate roles of the two recombinases is emerging.

Plasmid pSC101 harbors a recombination site, psi, which is able to resolve plasmid multimers and to substitute for the analogous chromosomal Escherichia coli site dif

Plasmid pSC101 harbors a 28-bp sequence which is homologous to dif, the target site of the XerC/XerD-dependent recombination system in Escherichia coli. Using a technique which allows very sensitive detection of plasmid loss, we show that recombination at this site, termed psi for pSC101 stabilized inheritance, causes a moderate increase in pSC101 stability. The role of the psi sequence in site-specific recombination has been explored in two other contexts. It was cloned in a derivative of plasmid p15A and inserted into the chromosome in place of dif. In the first situation, psi activity requires accessory sequences and results in multimer resolution; in the second situation, it suppresses the effects of the dif deletion and can promote intermolecular exchanges. Thus, psi is a site whose recombinational activity depends on the context, the first in the cer/dif family known to exhibit such flexibility.

Binding and transcriptional activation of the promoter for the neural cell adhesion molecule by HoxC6 (Hox-3.3).

Scores of homeobox gene-encoded transcription factors are expressed in a definite spatiotemporal pattern during embryogenesis and regulate a series of as yet unidentified target genes to help coordinate the morphogenetic process. We have suggested that homeobox gene products modulate the expression of adhesion molecule genes and have shown in cotransfection experiments that the promoters for the neural cell adhesion molecule (N-CAM) and cytotactin/tenascin genes respond to cues from different homeobox-containing genes. In this study, we show that the HoxC6 (Hox-3.3)-encoded homeoprotein binds to a DNA sequence in the N-CAM promoter CCTAATTATTAA, designated homeodomain binding site I (HBS-I). To test whether HoxC6 regulated N-CAM promoter activity, we cotransfected the Long and Short reading frame variants of Xenopus HoxC6 (CMV-HoxC6-L and CMV-HoxC6-S) driven by the human cytomegalovirus (CMV) promoter together with a chloramphenicol acetyltransferase (CAT) reporter gene driven by the mouse N-CAM promoter (N-CAM-Pro-CAT). Cotransfection of NIH 3T3 cells with either of the CMV-HoxC6 expression vectors stimulated N-CAM promoter-driven CAT expression. A 47-bp region from the N-CAM promoter that included HBS-I and an adjacent potential HBS, HBS-II, conferred HoxC6 regulation on a simian virus 40 minimal promoter. HBS-I was sufficient for transactivation of the minimal promoter by CMV-HoxC6-S. However, transcriptional activation by CMV-HoxC6-L required both HBS-I and HBS-II, inasmuch as mutation of either HBS-I, HBS-II, or both motifs abolished the response. These studies suggest that HBS-I is a target site for binding and transcriptional control of the N-CAM promoter by homeoproteins, although accessory DNA sequences (such as HBS-II) may also be required. Together with previous studies, these results support the notion that N-CAM gene expression may be controlled by different combinations of homeoproteins that appear in a place-dependent manner during embryogenesis.

Phenotype-associated env gene variation among eight related human immunodeficiency virus type 1 clones: evidence for in vivo recombination and determinants of cytotropism outside the V3 domain.

The nucleotide sequences of the env genes of eight phenotypically heterogeneous human immunodeficiency virus type 1 (HIV-1) clones recovered from a single individual within a 3-week period were compared. In addition, the accessory gene sequences for four of these clones were obtained. Variation among most accessory genes was limited. In contrast, pronounced phenotype-associated sequence variation was observed in the env gene. At least three of these clones most likely resulted from genetic recombination events in vivo, indicating that this phenomenon may account for the emergence of proviruses with novel phenotypic properties. Within the env genes of the eight clones, four domains could be defined, the sequence of each of which clustered in two groups with high internal homology but 11 to 30% cluster variation. The extensive env gene variation among these eight clones could largely be explained by the unique manner in which the alleles of these four domains were combined in each clone. Experiments with chimeric proviruses demonstrated that the HIV-1 env gene determined the capacity to induce syncytia and tropism for T-cell lines. Amino acids previously shown to be involved in gp120-CD4 and gp120-gp41 interaction were completely conserved among these eight clones. The finding of identical V3 sequences in clones differing in tropism for primary monocytes and T-cell lines demonstrated the existence of determinants of tropism outside the env V3 region.

Delimitation of cohesive ends site ( cos) of Streptomyces temperate bacteriophage R4

The cohesive ends site (cos) of actinophage R4 was delimitated by assaying the in vivo packaging activity of cosmid derivatives in Streptomyces lividans. A region of 66 bp from -30 to +36 from the center of cohesive ends was required for the basal level of packaging activity. Two additional regions outside the basal sequences from -39 to -31 and from +37 to +97 were necessary for the high level of activity, defined as the accessory sequences. Direct- or inverted-repeat sequences were found within the delimitated region, which might be involved in the recognition by the terminase of actinophage R4.

Creating phyllotaxis: the dislodgement model

Assuming that neither the Fibonacci sequence nor any numerical ratio or angular deflection is specified in the genetic material of a plant cell, there must be an arranging mechanism effecting the sequence mentioned. Considering the ubiquity of the Fibonacci numbers in nature, embracing many species of flora, we expect a very simple geometrical law to be responsible. Success in finding such a law does not constitute a proof, but it is at the least an indication that we should look here with mathematical rather than biological eyes. The idea may seem self-evident. However, in the literature it has not yet been honored as the basis for constructing the phyllotaxis in centric, planar models. It is shown here that for the construction of a phyllotactic structure, no special angles or distances need be defined; natural growth functions can be used; planar, cylindrical, conical, and paraboloid constructions are possible within the same model; and constructions leading to accessory sequences and multijugate sequences can also be carried out.

Specific accessory sequences in Saccharomyces cerevisiae introns control assembly of pre-mRNAs into spliceosomes.

In experiments involving deletion and rearrangement of intron sequences two small regions of the intron in the yeast CYH2 ribosomal protein gene were found to play important roles in splicing of the pre-mRNA. One element lies downstream of the 5' splice site, and the other is upstream of the branchpoint sequence UACUAAC. Deletion of the element upstream of the branchpoint prevents spliceosome formation and blocks splicing in vivo and in vitro. Deletion of the element downstream of the 5' splice site does not on its own block splicing but rescues spliceosome formation and splicing of pre-mRNA lacking the element upstream of the branchpoint. These elements correspond to two regions of sequence complementarity which are a conserved feature of the introns in yeast pre-mRNAs. Mixing and matching of the elements from the ACT1 and CYH2 gene introns showed that these elements can cooperate in an intron-specific fashion to control spliceosome assembly.

Adenovirus origin of DNA replication: sequence requirements for replication in vitro.

The initiation of adenovirus DNA takes place at the termini of the viral genome and requires the presence of specific nucleotide sequence elements. To define the sequence organization of the viral origin, we tested a large number of deletion, insertion, and base substitution mutants for their ability to support initiation and replication in vitro. The data demonstrate that the origin consists of at least three functionally distinct domains, A, B, and C. Domain A (nucleotides 1 to 18) contains the minimal sequence sufficient for origin function. Domains B (nucleotides 19 to 40) and C (nucleotides 41 to 51) contain accessory sequences that significantly increase the activity of the minimal origin. The presence of domain B increases the efficiency of initiation by more than 10-fold in vitro, and the presence of domains B and C increases the efficiency of initiation by more than 30-fold. Mutations that alter the distance between the minimal origin and the accessory domains by one or two base pairs dramatically decrease initiation efficiency. This critical spacing requirement suggests that there are specific interactions between the factors that recognize the two regions.

Adenovirus origin of DNA replication: sequence requirements for replication in vitro.

The initiation of adenovirus DNA takes place at the termini of the viral genome and requires the presence of specific nucleotide sequence elements. To define the sequence organization of the viral origin, we tested a large number of deletion, insertion, and base substitution mutants for their ability to support initiation and replication in vitro. The data demonstrate that the origin consists of at least three functionally distinct domains, A, B, and C. Domain A (nucleotides 1 to 18) contains the minimal sequence sufficient for origin function. Domains B (nucleotides 19 to 40) and C (nucleotides 41 to 51) contain accessory sequences that significantly increase the activity of the minimal origin. The presence of domain B increases the efficiency of initiation by more than 10-fold in vitro, and the presence of domains B and C increases the efficiency of initiation by more than 30-fold. Mutations that alter the distance between the minimal origin and the accessory domains by one or two base pairs dramatically decrease initiation efficiency. This critical spacing requirement suggests that there are specific interactions between the factors that recognize the two regions.