Conserved structural features are found upstream from the three co-ordinately regulated discoidin I genes of Dictyostelium discoideum

The discoidin I genes of Dictyostelium form a small, co-ordinately regulated multigene family. We have sequenced and compared the upstream regions of the DiscI-alpha, -beta and -gamma genes. For the most part the upstream regions of the three genes are non-homologous. The upstream sequences of the beta and gamma genes are exceedingly A + T-rich, while those of the alpha gene are less so. All three genes have a relatively G + C-rich region 20 to 40 base-pairs in length, found approximately 200 base-pairs 5' to the messenger RNA start site. This G + C-rich region 5' to the beta and gamma genes is flanked by short inverted repeats. Within this region, there is an 11 base-pair exact homology between the alpha and gamma genes, and a less perfect homology between these genes and the beta gene. The homology is flanked at a short distance by interspersed G and T residues. The gamma gene is greater than 90% A + T for greater than 800 base-pairs upstream. Further upstream there is a G + C-rich region that is also found inverted approximately 3.5 X 10(3) base-pairs away. The gamma and beta genes are tandemly linked, and the entire approximately 500 base-pair intergene region between the 3' end of the gamma gene and the 5' end of the beta gene is A + T-rich (approximately 90%) with the exception of the homology region 5' to the gamma gene. We demonstrate also the presence of a discoidin I pseudogene fragment having only 139 base-pairs of discoidin homology with greater than 8% mismatch. It is flanked upstream by five 39 base-pair G + C-rich repeats, and downstream by sequences that are extremely A + T-rich. We discuss the possible significance of the conserved G + C-rich structures on discoidin I gene expression.     

mRNA processing in Dictyostelium: sequence requirements for termination and splicing

Criteria for the identification of termination regions in Dictyostelium discoideum genes have been established and the sequence requirements for termination in 33 genes have been analyzed. A canonical hexamer signal AATAAA was present 15-30 nucleotides upstream of the cleavage site, usually a TA, and was embedded in a particularly A-rich environment. T- or GT-rich downstream elements characteristic of animal cells could not be identified. In a sample of 102 introns we have established the consensus AG/GTAAGT and ATAG/ for the 5' and 3' splice sites, respectively. Most introns are 75-150 nucleotides long and the A+T content is high (90%). A putative branch point was identified in half of the introns 20-60 nucleotides upstream of the 3' splice site and the consensus TACTAAY was derived. A polypyrimidine tract required for branching in vertebrates was not identified, but weak preference for pyrimidine was found 10-45 nucleotides upstream of the 3' splice site.     

Design and synthesis of specific inhibitors of the 3'-processing step of HIV-1 integrase

The novel dinucleotide 5'-phosphate, [(L,D)-pIsodApdC], discovered in our laboratory, is a strong inhibitor of HIV-1 integrase for both the 3'-processing and the strand transfer steps. The rationale used in this molecular design was that residues immediately upstream of the dinucleotide cleavage site in the 3'-processing step might provide critical recognition/binding sites on integrase. The rationale for the second type of inhibitors was based on the elimination products (linear and cyclic dinucleotides) of 3'-processing. However, while the linear dinucleotide 5'-phosphate (pdGpdT) was active, its cyclic counterpart was inactive against both wild-type and mutant HIV integrase.     

Interactions of the human T-cell leukemia virus type-II integrase with the conserved CA in the retroviral long terminal repeat end

Retroviral integrases (INs) interact with termini of retroviral DNA in the conserved 5'-C(A/G)T. For most integrases, modifications of critical moieties in the major and minor grooves of these sequences decrease 3'-processing. However, for human immunodeficiency virus type-2 (HTLV-2) IN, the replacement of the guanine with 6-methylguanine or hypoxanthine not only reduced 3'-processing, but also promoted cleavage at a second site. This novel cleavage activity required an upstream ACA, unique to the HTLV-2 U5 end. 3'-Processing assays with additional isosteric modifications at Gua and filter binding experiments revealed that the mechanism of the second site cleavage differed among the major groove, minor groove, and mismatch modifications. Importantly, the decrease in 3'-processing activity noted with the minor groove and mismatch modifications were attributed to a decrease in binding. Major groove modifications, however, decreased the level of 3'-processing, but did not affect binding. This suggests that integrase binds the viral end through the minor groove, but relies on major groove contacts for 3'-processing. Several modifications were also examined in strand transfer and disintegration substrates. HTLV-2 IN showed reduced activity with strand transfer and disintegration substrates containing major groove, but not minor groove modifications. This suggests major groove interactions at guanine also provide an important role in these reactions.     

High A + T content conserved in DNA sequences upstream of leuABCD in Escherichia coli and Salmonella typhimurium.

The nucleotide sequence of over 800 base pairs of DNA upstream of leuP was determined for Escherichia coli and Salmonella typhimurium. In both of these enteric bacteria, approximately 500 base pairs of A + T-rich sequences separates leuP from an upstream open reading frame. Although these A + T-rich sequences share little homology, the distribution of A + T base pairs within the region is strikingly conserved. Deletion of the A + T-rich sequences upstream of the E. coli leu operon does not markedly affect the strength of the leu promoter in vivo.