NXT1 (p15) is a crucial cellular cofactor in TAP-dependent export of intron-containing RNA in mammalian cells.

TAP, the human homologue of the yeast protein Mex67p, has been proposed to serve a role in mRNA export in mammalian cells. We have examined the ability of TAP to mediate export of Rev response element (RRE)-containing human immunodeficiency virus (HIV) RNA, a well-characterized export substrate in mammalian cells. To do this, the TAP gene was fused in frame to either RevM10 or RevDelta78-79. These proteins are nonfunctional Rev mutant proteins that can bind to HIV RNA containing the RRE in vivo but are unable to mediate the export of this RNA to the cytoplasm. However, the fusion of TAP to either of these mutant proteins gave rise to chimeric proteins that were able to complement Rev function. Significantly, cotransfection with a vector expressing NXT1 (p15), an NTF2-related cellular factor that binds to TAP, led to dramatic enhancement of the ability of the chimeric proteins to mediate RNA export. Mutant-protein analysis demonstrated that the domain necessary for nuclear export mapped to the C-terminal region of TAP and required the domain that interacts with NXT1, as well as the region that has been shown to interact with nucleoporins. RevM10-TAP function was leptomycin B insensitive. In contrast, the function of this protein was inhibited by DeltaCAN, a protein consisting of part of the FG repeat domain of CAN/Nup214. These results show that TAP can complement Rev nuclear export signal function and redirect the export of intron-containing RNA to a CRM1-independent pathway. These experiments support the role of TAP as an RNA export factor in mammalian cells. In addition, they indicate that NXT1 serves as a crucial cellular cofactor in this process.

Molecular cloning and functional expression in lactobacillus plantarum 80 of xylT, encoding the D-xylose-H+ symporter of Lactobacillus brevis.

A 3-kb region, located downstream of the Lactobacillus brevis xylA gene (encoding D-xylose isomerase), was cloned in Escherichia coli TG1. The sequence revealed two open reading frames which could code for the D-xylulose kinase gene (xylB) and another gene (xylT) encoding a protein of 457 amino acids with significant similarity to the D-xylose-H+ symporters of E. coli, XylE (57%), and Bacillus megaterium, XylT (58%), to the D-xylose-Na+ symporter of Tetragenococcus halophila, XylE (57%), and to the L-arabinose-H+ symporter of E. coli, AraE (60%). The L. brevis xylABT genes showed an arrangement similar to that of the B. megaterium xylABT operon and the T. halophila xylABE operon. Southern hybridization performed with the Lactobacillus pentosus xylR gene (encoding the D-xylose repressor protein) as a probe revealed the existence of a xylR homologue in L. brevis which is not located with the xyABT locus. The existence of a functional XylR was further suggested by the presence of xylO sequences upstream of xylA and xylT and by the requirement of D-xylose for the induction of D-xylose isomerase, D-xylulose kinase, and D-xylose transport activities in L. brevis. When L. brevis was cultivated in a mixture of D-glucose and D-xylose, the D-xylose isomerase and D-xylulose kinase activities were reduced fourfold and the D-xylose transport activity was reduced by sixfold, suggesting catabolite repression by D-glucose of D-xylose assimilation. The xylT gene was functionally expressed in Lactobacillus plantarum 80, a strain which lacks proton motive force-linked D-xylose transport activity. The role of the XylT protein was confirmed by the accumulation of D-xylose in L. plantarum 80 cells, and this accumulation was dependent on the proton motive force generated by either malolactic fermentation or by the metabolism of D-glucose. The apparent affinity constant of XylT for D-xylose was approximately 215 microM, and the maximal initial velocity of transport was 35 nmol/min per mg (dry weight). Furthermore, of a number of sugars tested, only 6-deoxy-D-glucose inhibited the transport of D-xylose by XylT competitively, with a Ki of 220 microM.

Target-sequence preferences of HIV-1 integration complexes in vitro.

Integration of reverse transcribed retroviral cDNA is not restricted to particular host DNA sequences. However, the frequency of integration into a particular phosphodiester bond is influenced by the local sequence. Here we examine the target-sequence preferences of purified HIV integrase and viral nucleoprotein complexes (preintegration complexes) isolated from freshly infected cells. We find that the three-base sequence including the integration site is not the major factor determining the frequency of integration, since identical triplets embedded in different sequences are used with very different efficiencies. However, there is a statistically significant bias against integration upstream of a pyrimidine nucleotide. The target-sequence preferences of purified integrase and preintegration complexes are very different. Strong integration sites on opposite DNA strands occur in pairs separated by five residues when preintegration complexes are used but not with purified integrase. These studies highlight the difference between the two sources of HIV integration activity and may provide the basis for a simple assay for the correct assembly of viral nucleoprotein complexes.

In vitro integration of human immunodeficiency virus type 1 cDNA into targets containing protein-induced bends.

Integration of human immunodeficiency virus type 1 cDNA into a target DNA can be strongly influenced by the conformation of the target. For example, integration in vitro is sometimes favored in target DNAs containing sequence-directed bends or DNA distortions caused by bound proteins. We have analyzed the effect of DNA bending by studying integration into two well-characterized protein-DNA complexes: Escherichia coli integration host factor (IHF) protein bound to a phage IHF site, and the DNA binding domain of human lymphoid enhancer factor (LEF) bound to a LEF site. Both of these proteins have previously been reported to bend DNA by approximately 140 degrees. Binding of IHF greatly increases the efficiency of in vitro integration at hotspots within the IHF site. We analyzed a series of mutants in which the IHF site was modified at the most prominent hotspot. We found that each variant still displayed enhanced integration upon IHF binding. Evidently the local sequence is not critical for formation of an IHF hotspot. LEF binding did not create preferred sites for integration. The different effects of IHF and LEF binding can be rationalized in terms of the different proposed conformations of the two protein-DNA complexes.

Target DNA capture by HIV-1 integration complexes.

BACKGROUND: The early steps of human immunodeficiency virus 1 (HIV-1) replication involve reverse transcription of the viral RNA and integration of the resulting cDNA into a host chromosome. The DNA integration step requires the integration machinery ('preintegration complex') to bind to the host DNA before connecting the viral and host DNAs. Here, we present experiments that distinguish among three possible pathways of target-DNA capture: repeated binding and release of target DNA prior to the chemical strand-transfer step; binding followed by facilitated diffusion along target DNA (sliding); and integration at the initial target-capture site. The mechanism of target-DNA capture has implications for the design of gene therapy methods, and influences the interpretation of results on the selection of integration target sites in vivo. RESULTS: We present new in vitro conditions that allow us to assemble HIV-1 integrase--the virus-encoded recombination enzyme--with a viral DNA and then to trap assembled complexes bound to target DNA. We find that complexes of integrase and viral DNA do not slide along target DNA substantially after binding. We confirm and extend these results by analyzing target capture by a hybrid protein composed of HIV-1 integrase linked to a sequence-specific DNA-binding domain. We find that the integrase domain binds quickly and tightly under the above conditions, thereby obstructing function of the fused sequence-specific DNA-binding domain. We also monitor target-DNA capture by HIV-1 preintegration complexes purified from freshly infected cells. Partially purified complexes commit quickly and stably to the first target DNA added, whereas preintegration complexes in crude cytoplasmic extracts do not. The addition of extracts from uninfected cells to partially purified complexes blocks quick commitment. CONCLUSIONS: Under new conditions favorable for the analysis of target-DNA capture in vitro, HIV-1 integrase complexes bind quickly and stably to target DNA without subsequent sliding. Parallel studies of preintegration complexes support a model in which target-site capture in vivo is reversible as a result of the action of cellular factors.

Use of antisense RNA to confer bacteriophage resistance in dairy starter cultures.

The strategy and implementation of a unique system for engineering bacteriophage resistant starter cultures of Lactococcus lactis employing antisense RNA is reviewed. As a necessary prerequisite for developing this system, we have cloned and sequenced a number of bacteriophage genes coding for minor and major structural proteins. In addition, we have also identified a series of genes whose function(s) is not known but their sequences appear to be conserved in a vast number of isolates. One of these latter sequences, designated gp51C, codes for a 51-kDa protein which is extremely charged and shares some homology with yeast translation initiation factor. Resistance to a broad class of isometric bacteriophages has been achieved by expression of an antisense RNA targeted against, for example, gp51C. In the best case, expression of the antisense gp51C RNA results is a greater than 99% reduction in the total number of plaque forming units. Additional antisense RNA constructs directed against other bacteriophage genes, including the major capsid protein, also appear effective at inhibiting infection from 40-55% suggesting that this approach may prove useful for engineering a set of truly isogenic strains to be used in a starter culture rotation plan.

Bacteriophage resistance in Lactococcus lactis ssp. lactis using antisense ribonucleic acid.

Antisense RNA against a conserved bacteriophage gene when expressed in a Lactococcus lactis ssp. lactis strain renders it resistant to bacteriophage infection. Two open reading frames have been identified in a L. lactis ssp. lactis bacteriophage that are conserved in a majority of isolates. They code for an 18-kDa (designated GP18C) protein and a 24-kDa (GP24C) protein, respectively, which are arranged along with previously identified open reading frames in a tandem motif similar to other bacteriophages. The presence of gp18C and gp24C in a number of bacteriophage isolates was confirmed by polymerase chain reaction using primers specific for these regions. Plasmids bearing various fragments of gp18C, gp24C, or both were constructed such that the respective open reading frames were positioned in the antisense direction relative to the Lactococcus lactis ssp. cremoris Wg2 promoter, p59. These antisense RNA-producing vectors inhibited the efficiency of plaquing of L. lactis ssp. lactis bacteriophage phi 7-9 up to 50%; the resulting plaques were extremely small and irregular in shape. The replication of the bacteriophage was severely inhibited, and the total number decreased over the first 3 h during infection in strains expressing antisense RNA compared with the host strain alone, in which the bacteriophage number increased 10(4)-fold.

Cloning and sequencing the Lactobacillus brevis gene encoding xylose isomerase.

The gene (xylA) coding for the Lactobacillus brevis xylose isomerase (Xi) has been isolated and its complete nucleotide sequence determined. L. brevis Xi was purified and the N-terminal sequence determined. All attempts to directly clone the intact xylA using a degenerative primer deduced from amino acids (aa) 10-14 were not successful. A fragment coding for the first 462 bp from the 5' end of xylA was isolated by PCR with two primers, one coding for aa M36 to W43 and the second coding for an aa sequence (WGGREG) conserved in a number of Xi's isolated from other bacteria. From the sequence of this fragment, two additional PCR primers were synthesized, which were used in an 'outward' reaction to clone a 546-bp fragment including a region upstream from the N terminus. Finally, the complete xylA gene was cloned in a 0.43-kb NlaIII-SalI fragment and a 1.9-kb SalI-EcoRI fragment. The 449-aa sequence for the L. brevis Xi shows homology with Xis isolated from other bacteria, especially within the primary catalytic domains of the enzyme.