pA Sites Research Articles

Retroviral vectors produced in the cytoplasmic vaccinia virus system transduce intron-containing genes.

Introns and polyadenylation (pA) sites are known to improve transcript stability and nuclear-cytoplasmic transport and are normally present in efficient gene expression vectors. Standard retroviral vectors, however, do not allow the inclusion of such sequence elements, as mRNA processing at internal splice and pA sites interferes with the production of functional full-length vector genomes. In this report we examined the capability of hybrid vaccinia/retroviral vectors to transduce complex gene cassettes with nuclear RNA processing signals within the retroviral genome. A retroviral vector was constructed that contains a gene of interest (the human coagulation factor IX [FIX] cDNA), including an intron and an internal pA site. The modified proviral vector genome was cloned downstream of a vaccinia virus promoter and was inserted into the vaccinia virus genome. Infection of a packaging cell line with the recombinant vaccinia virus vector resulted in secretion of retroviral particles at average titers of 10(5) CFU per ml of cell culture supernatant. Due to the cytoplasmic transcription and the nonrecognition of nuclear transcription signals in the vaccinia virus system, full-length transcripts were obtained that still contained the intron. In the retrovirally transduced cell lines the FIX transcripts were terminated at the internal pA site. The transcripts were quantitatively spliced, and FIX was secreted. Recombinant cell lines with stable single-copy inserts containing sequence elements necessary for efficient gene function could be generated. Thus, a relatively simple cytoplasmic system for the generation of complex retroviral vectors is described. Retroviral vectors transducing intron-containing gene cassettes may play a further role in gene therapy applications.

Extensive Combinations of Clustering Sets of Sulfuric Acid and Various Solvents.

The FAB mass spectra of the mixtures of the sulfuric acid and 14 solvent molecules (acetone, methyl alcohol, ethyl alcohol, 1-propyl alcohol, 2-propyl alcohol, 1-butyl alcohol, 2-butyl alcohol, t-butyl alcohol, 1-pentyl alcohol, 1-hexyl alcohol, ethylene glycol, acetic acid, dimethyl sulfoxide, and water) were measured and CAD spectra of the cluster ions were also measured.The FAB mass spectra showed the extensive cluster ions of H2SO4 with the solvent, (aH2SO4+bSolv+H)+, a and b are positive integers or zero. We also tried to investigate CAD spectra of unsymmetrical protonated ions of sulfuric acid and water molecules, (H2SO4+H2O+H)+, precisely.The mass spectra were found to be dehydrated ions, (aH2SO4+bSolv-H2O+H)+. In some cases, we determined which dehydration occurred from H2SO4 site or from solvent site. For that problem to be solved, the CAD spectra by means of MS/MS (mass spectrometry/mass spectrometry, tandem mass spectrometry) of (aH2SO4+bSolv+H)+ and (aH2SO4+bSolv-H2O+H)+, were needed to measure. Further the special cluster ions (CnH2n+1OH-H2O+H)+, if proton resides on the larger PA site, the dehydration occurs on the protonated site. We can decide the dehydration site by the present methods.

Regulation of polyadenylation in hepatitis B viruses: stimulation by the upstream activating signal PS1 is orientation-dependent, distance-independent, and additive

Hepatitis B viruses replicate by reverse transcription of a genomic RNA which harbors terminal redundancies. The synthesis of this RNA requires that transcription proceed twice through the polyadenylation (pA) site which, in mammalian strains, is flanked by the variant hexanucleotide UAUAAA and a T-rich downstream domain. These core elements are by themselves virtually defective in 3' end processing and require multiple upstream accessory elements which regulate pA site use. In ground squirrel hepatitis B virus (GSHV), one of these signals (PS1; -215 to -107 relative to UAUAAA) is transcribed only at the 3' end of genomic RNA and as such is analogous to retroviral U3 sequences. PS1 cooperates with other signals to enhance pA site use to very high levels and can be further sub-divided into two regions (A and B) which contribute equally to 3' end processing. Critical residues within PS1B have been localized to a 15 bp A/T-rich stretch which displays homology to other known upstream activating signals. A 15 bp segment within PS1A which has the identical A/T content but a divergent primary sequence plays a diminished role in processing. Furthermore, PS1 can activate GSHV core element usage autonomously. This stimulation has been shown to be additive since multiple copies of PS1 progressively increase polyadenylation, a phenomenon which also demands that PS1 exert its influence from a variety of distances from the hexanucleotide signal.

Analysis of polyadenylation site usage of the c-myc oncogene.

The c-myc gene contains 2 well conserved polyadenylation (pA) sites. In all human and rat cell lines from various differentiation stages and tissue types the amount of mRNA terminating at the second pA site is 6-fold higher than the amount ending at the upstream site. This is not due to a difference in stability of the two mRNA types and therefore must be due to preferential usage of the downstream site. The usage of the pA sites is not altered during growth factor induction of quiescent cells. We have not been able to detect differences in behavior between mRNAs ending at either pA site. Both types of mRNA are induced upon treatment of cells with cycloheximide. Furthermore, we have shown that the poly(A) tail of c-myc mRNA is lost during degradation of the messenger, as was described previously for c-myc mRNA in an in vitro system. The time required for the loss of the poly(A) tail is similar to the half-life of c-myc mRNA.

New data from peat bogs may give a historical perspective on acid deposition

Dynamic changes in certain bog ecosystems together with environmental trace elements may be utilized to reconstruct the history of atmospheric chemicals deposited on the terrestrial biosphere. Cores collected from ombrotrophic bogs in eastern New York, western Pennsylvania and western Virginia were dated using the Pb-210 method and analyzed for 34 elements by neutron activation analysis. The fallout Cs-137 profiles were utilized to cross check the Pb-210 dates assigned for each layer and to estimate the transport of mobile elements similar to Cs. A comparison has been made between the relative enrichment factors of the elements accumulated at different times since 1800 on the dated peat bog layers and the crustal abundance of elements compared to Al. At the PA site, relative enrichments of greater than 100 times are found for C1, N, S and Br; relative enrichments of 10 to 40 times are found for Pb, Ca and Sb. At the NY site, generally lower relative enrichments are found with values of 10 to 40 times for C1, Cr, and Mn and less than 10 times for N and S.