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J J Rossi

Publications and source records attributed to J J Rossi.

At least 19 recordsLinked to original sources
PubMed1992

Small sequence insertions within the branch point region dictate alternative sites of lariat formation in a yeast intron.

The problem of intron recognition in S. cerevisiae appears to be in part solved by the strong conservation of intron encoded splicing signals, in particular the 5' GUAUGU and the branch point UACUAAC which interact via base pairing with the RNA components of U1 and U2 snRNPs respectively. Nevertheless, the mere presence of such signals is insufficient for splicing to occur. In the S. cerevisiae ACT1 intron, a silent UACUAAC-like sequence (UACUAAG) is located 7 nucleotides upstream of the canonical branch point signal. In order to investigate whether other factors, in addition to the U2-UACUAAC base-pair interactions, affect branch point selection in yeast, we created a cis-competition assay by converting the UACUAAG to a strong branch point signal (UACUAAC). If simply having a canonical UACUAAC sequence were sufficient for lariat formation, a 1:1 ratio in usage of the two signals should have been observed. In this double branch point intron, however, the downstream UACUAAC is utilized preferentially (4:1). Results obtained from the analyses of numerous sequence variants flanking the two UACUAAC sequences, demonstrate that non-conserved sequences in the branch point region are able to define lariat formation. Consequently, we conclude that U2 base-pairing is not the only requirement determining branch point selection in yeast, and local structure in the vicinity of the branch point could play a critical role in its recognition.

Alternative Splicing↗
PubMed1992

Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.

Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents.

Amino Acid Sequence↗
PubMed1992

Differential transcription of Pgk genes during spermatogenesis in the mouse.

We have analyzed the occurrence of transcripts produced from the ubiquitously expressed, X-linked Pgk-1 gene and the testis-specific, autosomal Pgk-2 gene during spermatogenesis in the mouse. We found that tissue specificity, developmental specificity, and cell-type specificity of these mRNAs parallel that previously reported for the two protein isozymes of phosphoglycerate kinase (PGK) encoded by these two genes. This indicates that primary regulation of differential expression of the Pgk genes during spermatogenesis is exerted at the transcriptional level. We first detected Pgk-2 mRNA in preleptotene spermatocytes, indicating that transcription of Pgk-2 is initiated coincident with the onset of meiosis in male germ cells, and then continues to increase in later spermatocytes and postmeiotic round spermatids. This expression initiates prior to an initial decline in Pgk-1 transcript levels observed in pachytene spermatocytes, which apparently follows inactivation of the single X chromosome in spermatogenic cells. However, unlike cessation of Pgk-1 transcription from the inactivated X chromosome in female somatic cells, we show that inactivation of the Pgk-1 locus in spermatogenic cells is not followed by methylation of a key CpG dinucleotide in the promoter region. These results support the idea that specific expression of the Pgk-2 gene in meiotic and postmeiotic spermatogenic cells has evolved to compensate for reduced levels of Pgk-1 gene product caused by transient X-chromosome inactivation in these cells. They further suggest that reinitiation of transcription of the paternal Pgk-1 allele shortly after fertilization is facilitated by constitutive hypomethylation in the promoter region of this gene throughout spermatogenesis.

Animals↗
PubMed1992

Ribozymes.

RNA enzymes or ribozymes are receiving considerable attention for their potential use as highly specific inhibitors of gene expression. From the basic science perspective, the mechanisms by which ribozymes catalyze site-specific cleavage (and in some cases ligation) reactions provide exciting and active areas of scientific investigation. The most recent developments in our understanding of the molecular mechanisms of catalysis, as well as in vivo applications of ribozymes, are highlighted.

Animals↗
PubMed1992

PCR-based quantitation of low levels of HIV-1 DNA by using an external standard.

Beginning with 10(3)-10(5) molecules of a purified HIV-1 target sequence as a starting template, we have examined the effects of starting template concentration and cycle number on the amplification efficiency of the polymerase chain reaction. An external standard DNA sequence has been designed that when added to a DNA sample enables a determination of the starting concentration of HIV-1 target sequence in that sample of DNA. Varying ratios of external standard and target DNA sequences were amplified for 22 cycles. When the starting concentration of the external standard was within 50-fold of the starting concentration of the target, the amplifications of both sequences were proportional. These same results were obtained when the two templates were amplified in the presence of an excess of heterogeneous genomic DNA. Using this quantitative method, the number of starting target molecules in a DNA sample can be calculated to within a two-fold range of accuracy.

Base Sequence↗
PubMed1992

Ribozymes as anti-HIV-1 therapeutic agents: principles, applications, and problems.

An emerging strategy in the treatment of viral infections is the use of antisense DNA or RNA to pair with, and block expression of viral transcripts. RNA, in addition to being an informational molecule, can also possess enzymatic activity. Thus, by combining anti-sense and enzymatic functions into a single transcript, it is now possible to design catalytic RNAs, or ribozymes, which can specifically pair with virtually any viral RNA, and cleave the phosphodiester backbone at a specified location, thereby functionally inactivating the viral RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. There are several different catalytic motifs which possess enzymatic activity, and each one of these can be incorporated into an enzymatic antisense with site-specific cleavage capabilities. By focusing on one type of catalytic motif, the hammerhead, we describe the principles behind the development of ribozymes as transacting, site-specific ribonucleases, several applications of ribozymes in functional destruction of target RNAs, as well as several of the problems confronting their use. We also describe a liposome delivery system which facilitates intracellular inclusion of ribozymes, and may provide a means for therapeutic delivery of ribozymes to HIV-1 infected cells.

Animals↗
PubMed1992

Substitution of non-catalytic stem and loop regions of hammerhead ribozyme with DNA counterparts only increases KM without sacrificing the catalytic step (kcat): a way to improve substrate-specificity.

In elucidating structure-function relationships and stabilizing ribozymes in vivo, several chimeric RNA/DNA ribozymes and substrates were chemically synthesized. Measurements of kinetic parameters revealed that the maximally deoxyribonucleotide-substituted ribozyme (DRDRD32) gained the highest catalytic activity reaching the kcat value of > 10 min-1, the highest value ever reported for hammerhead-type ribozymes. Since these chimeric ribozymes are more stable than the wild-type all-RNA ribozymes in vivo and they also possess higher substrate-specificity, they are considered to be better candidates for antiviral therapeutic agents.

Base Sequence↗
PubMed1992

Biological and functional aspects of catalytic RNAs.

Several naturally occurring ribozymes have now been well characterized with respect to their in vivo and in vitro activities. Through detailed biochemical and genetic analyses, it has become possible to alter the substrate specificity of each ribozyme using simple Watson-Crick base pairing. Several laboratories, therefore, have designed ribozymes to cleave viral or other cellular transcripts in vitro with the hope of developing these molecules as antiviral or therapeutic agents. In addition to Watson-Crick base pairing, however, other factors such as protein or RNA tertiary interactions are involved in the ribozyme cleavage activity. Although several engineered ribozymes have been used successfully to reduce gene expression in vivo, it is difficult to determine whether gene expression has been reduced by the cleaving activity of the ribozyme or by its inherent antisense activity. In order to discriminate between these two activities and optimize potentially therapeutic ribozymes, it is imperative to develop in vivo assays in which the antisense activity of ribozymes is negligible.

Animals↗
PubMed1991

Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein.

The c-fos gene product Fos has been implicated in many cellular processes, including signal transduction, DNA synthesis, and resistance to antineoplastic agents. A fos ribozyme (catalytic RNA) was designed to evaluate the effects of suppressing Fos protein synthesis on expression of enzymes involved in DNA synthesis, DNA repair, and drug resistance. DNA encoding the fos ribozyme (fosRb) was cloned into the pMAMneo expression plasmid, and the resultant vector was transfected into A2780DDP cells resistant to the chemotherapeutic agent cisplatin. The parental drug-sensitive A2780S cells were transfected with the pMMV vector containing the c-fos gene. Morphological alterations were accompanied by significant changes in pharmacological sensitivity in both c-fos- and fosRb-transfected cells. pMAMneo fosRb transfectants revealed decreased c-fos gene expression, concomitant with reduced thymidylate (dTMP) synthase, DNA polymerase beta, topoisomerase I, and metallothionein IIA mRNAs. In contrast, c-myc expression was elevated after fos ribozyme action. Insertion of a mutant ribozyme, mainly capable of antisense activity, into A2780DDP cells resulted in smaller reductions in c-fos gene expression and in cisplatin resistance than the active ribozyme. These studies establish a role for c-fos in drug resistance and in mediating DNA synthesis and repair processes by modulating expression of genes such as dTMP synthase, DNA polymerase beta, and topoisomerase I. These studies also suggest the utility of ribozymes in the analysis of cellular gene expression.

Base Sequence↗
PubMed1991

Altered response to growth rate changes in Kluyveromyces lactis versus Saccharomyces cerevisiae as demonstrated by heterologous expression of ribosomal protein 59 (CRY1)

We report the cloning, characterization and preliminary analysis of the regulation of the gene coding for ribosomal protein 59 (RP59) from the budding yeast Kluyveromyces lactis. The RP59 gene is present as a single copy, contains an intron within the amino terminal coding portion of the gene, and harbors conserved S. cerevisiae splicing signals. Sequence elements upstream of the transcriptional start site are homologous to UASRPG, known to regulate the transcription of numerous genes in S. cerevisiae via their interaction with the trans-activating factor RAP1. These elements are necessary for transcription of RP59 in both K.lactis and S.cerevisiae hosts. UASRPG in S.cerevisiae rp genes also modulate the transcription of rp RNA synthesis in response to a growth rate upshift. In K.lactis, the RP59 gene does not respond to growth rate upshift. Reciprocal expression of RP59 and CRY1 in heterologous hosts demonstrates that glucose upshift occurs in S.cerevisiae but not K.lactis. These results demonstrate that a factor or factors required for growth upshift are lacking in K.lactis, and provide further evidence that the UASRPG are sufficient signals for modulating this response.

Base Sequence↗
PubMed1991

The potential use of catalytic RNAs in therapy of HIV infection and other diseases.

This article describes the applications (both real and potential) of a new antiviral strategy, based on the use of antisense, catalytic RNAs (ribozymes) as therapeutic agents. An understanding of both antisense inhibition of gene expression and RNA autocleavage reactions are essential to the use of this technology. In addition, for the successful application of this technology in clinical settings, an interdisciplinary approach involving clinicians, molecular and cellular biologists, will be necessary. The following treatise will highlight several salient features of ribozyme technology, emphasizing its application as an antiviral as well as discuss some problems and potential solutions pertinent to the clinical application of this technology.

Base Sequence↗
PubMed1991

Ribozyme-mediated cleavage of an HIV-1 gag RNA: the effects of nontargeted sequences and secondary structure on ribozyme cleavage activity.

Catalytic antisense RNAs, or ribozymes, have great potential as inhibitors of gene expression and as antiviral therapeutic agents. The major advantage of ribozymes versus standard antisense RNAs is their catalytic capability, enabling these RNAs to cleave multiple substrates. We have been investigating the antiviral activity of ribozymes targeted to the HIV-1 genome. The successful use of these antisense agents in an intracellular milieu requires stabilization of the ribozymes by flanking, non-base-pairing sequences, or some modification of the sugar-phosphate backbone. We describe a systematic investigation of the effects of flanking, non-base-pairing sequences on the catalytic activity of an anti-HIV-1 gag ribozyme embedded in radically different transcripts. Amazingly, these complex ribozyme-containing transcripts maintain substantial catalytic activity. Finally, we describe a bacterial gene fusion system that has potential for the large scale production of catalytically active ribozymes.

Base Sequence↗
PubMed1991

Persistence of bcr-able gene expression following bone marrow transplantation for chronic myelogenous leukemia in chronic phase.

The bcr-abl RNA transcript is the molecular counterpart of the Philadelphia chromosome and is detectable by an extremely sensitive polymerase chain reaction assay in most patients with chronic myelogenous leukemia. To determine the effectiveness of ablative radiochemotherapy and bone marrow transplantation in eradicating molecular evidence of the malignant clone, we assayed for bcr-abl RNA expression in specimens from 19 patients with CML in chronic phase (CP) who have survived for at least one year post-BMT. We correlated these results with the patients' remission status based on cytogenetic analysis and BM morphology, and with evidence of mixed hematopoietic chimerism by analysis of RBC antigen and DNA restriction fragment length polymorphism patterns. Thirteen of the 19 patients had detectable bcr-abl RNA at some time following BMT. Twelve of these patients have remained in remission by morphologic and karyotypic criteria from 16.6 to 63.7 months following BMT. One of these 13 patients relapsed both by cytogenetic and clinical criteria at 28.1 months after BMT. Six of these 13 patients are still positive at the time of their most recent analysis. Only two patients have evidence for mixed chimerism of normal hematopoietic elements by either RBC antigen or DNA RFLP patterns. These results suggest that, in some patients transplanted for CML in CP, small numbers of residual leukemic cells may persist or reappear transiently without leading to clinical relapse. The definition of complete remission in CML may need to be revised in light of the enhanced ability to detect minimal residual disease by PCR technology.

Adult↗
PubMed1991

Unexpected point mutations activate cryptic 3' splice sites by perturbing a natural secondary structure within a yeast intron.

The 3' splice site of the budding yeast Kluyveromyces lactis actin gene (ACT) intron is distally spaced (122 nucleotides) from its branchpoint and is also preceded by a silent PyAG located 43 nucleotides upstream. We devised a genetic screen that resulted in the isolation of several randomly induced cis-acting mutations that activate the silent PyAG as a 3' splice site. These mutations fall within a region surrounding this PyAG, which can hypothetically fold into a higher-order structure. Site-directed mutational analyses demonstrate that a hairpin structure in this region is required for correct 3' splice-site selection. Analysis of the point mutations suggests that local breathing of the hairpin near the first PyAG can lead to its activation. These data demonstrate that 3' splice-site selection is not a consequence of a linear, directional scanning mechanism, but support the notion of a critical positioning requirement for 3' splice-site selection. We speculate on the possible origin of this intron-encoded structural motif, which has homology to a bacterial transposon and suggests one possible origin for alternative splicing mechanisms in higher eukaryotes.

Base Composition↗
PubMed1991

Exploring the use of antisense, enzymatic RNA molecules (ribozymes) as therapeutic agents.

Antisense catalytic RNAs that specifically base-pair with and cleave target RNA sequences have potential for use as therapeutic agents against viral as well as endogenous gene expression. With the ultimate goal of developing anti-human immunodeficiency virus type 1 (HIV-1) ribozymes for therapeutic use, we have been exploring ways to improve upon the functional activity of ribozymes in living cells. This is being done by the systematic exploration of parameters that affect antisense, and hence ribozyme, function. These include target accessibility, stability of the catalyst, methods for delivery, and intracellular localization of the ribozyme. In addition, we have been examining the kinetic consequences of having extra, nontargeted sequences appended to the ribozyme flanking sequences. Perhaps the single most important consideration for ribozyme effectiveness in an intracellular environment is the accessibility of the target RNA for cleavage. By exploiting the mechanisms by which naturally occurring antisense RNAs interact with their target sequences, we hope to be able to address this problem of targeting and fully capitalize upon the potential of ribozymes as therapeutic agents.

Antiviral Agents↗