A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae. Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trp1, leu2 and ura3 mutations. A set of YCp and YIp vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT. These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HIS3, TRP1, LEU2 and URA3). They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well. Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast.

/pdf/a-system-of-shuttle-vectors-and-yeast-host-strains-designed-3bj6rqu23b.pdf

A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.

Vectors derived from human immunodeficiency virus (HIV) are highly efficient vehicles for in vivo gene delivery. However, their biosafety is of major concern. Here we exploit the complexity of the HIV genome to provide lentivirus vectors with novel biosafety features. In addition to the structural genes, HIV contains two regulatory genes, tat and rev, that are essential for HIV replication, and four accessory genes that encode critical virulence factors. We previously reported that the HIV type 1 accessory open reading frames are dispensable for efficient gene transduction by a lentivirus vector. We now demonstrate that the requirement for the tat gene can be offset by placing constitutive promoters upstream of the vector transcript. Vectors generated from constructs containing such a chimeric long terminal repeat (LTR) transduced neurons in vivo at very high efficiency, whether or not they were produced in the presence of Tat. When the rev gene was also deleted from the packaging construct, expression of gag and pol was strictly dependent on Rev complementation in trans. By the combined use of a separate nonoverlapping Rev expression plasmid and a 5' LTR chimeric transfer construct, we achieved optimal yields of vector of high transducing efficiency (up to 10(7) transducing units [TU]/ml and 10(4) TU/ng of p24). This third-generation lentivirus vector uses only a fractional set of HIV genes: gag, pol, and rev. Moreover, the HIV-derived constructs, and any recombinant between them, are contingent on upstream elements and trans complementation for expression and thus are nonfunctional outside of the vector producer cells. This split-genome, conditional packaging system is based on existing viral sequences and acts as a built-in device against the generation of productive recombinants. While the actual biosafety of the vector will ultimately be proven in vivo, the improved design presented here should facilitate testing of lentivirus vectors.

A Third-Generation Lentivirus Vector with a Conditional Packaging System

Viral replication usually requires that innate intracellular lines of defence be overcome, a task usually accomplished by specialized viral gene products The virion infectivity factor (Vif) protein of human immunodeficiency virus (HIV) is required during the late stages of viral production to counter the antiviral activity of APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; also known as CEM15), a protein expressed notably in human T lymphocytes When produced in the presence of APOBEC3G, vif-defective virus is non-infectious APOBEC3G is closely related to APOBEC1, the central component of an RNA-editing complex that deaminates a cytosine residue in apoB messenger RNA APOBEC family members also have potent DNA mutator activity through dC deamination; however, whether the editing potential of APOBEC3G has any relevance to HIV inhibition is unknown Here, we demonstrate that it does, as APOBEC3G exerts its antiviral effect during reverse transcription to trigger G-to-A hypermutation in the nascent retroviral DNA We also find that APOBEC3G can act on a broad range of retroviruses in addition to HIV, suggesting that hypermutation by editing is a general innate defence mechanism against this important group of pathogens

Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts

Considered by some to be among the simpler forms of life, viruses represent highly evolved natural vectors for the transfer of foreign genetic information into cells. This attribute has led to extensive attempts to engineer recombinant viral vectors for the delivery of therapeutic genes into diseased tissues. While substantial progress has been made, and some clinical successes are over the horizon, further vector refinement and/or development is required before gene therapy will become standard care for any individual disorder.

/pdf/viral-vectors-for-gene-therapy-the-art-of-turning-infectious-3minu6de1n.pdf

Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics

The position of the yeast phosphoglycerate kinase (PGK) gene has been mapped on a 2.95kb Hind III fragment. We have determined the nucleotide sequence of the 5' flanking region and compared this sequence with those from 16 other yeast genes. PGK, like all other yeast genes has an adenine residue at position -3. It has two possible TATA boxes at positions -114 and -152 and a CAAT box at -129. In addition we have defined a structure at position -63 to -39 that is common to all yeast genes that encode an abundant RNA. This structure is a CT-rich block followed, about 10 nucleotides later, by the sequence CAAG.

/pdf/conservation-of-high-efficiency-promoter-sequences-in-40x2oydy4y.pdf

Conservation of high efficiency promoter sequences in Saccharomyces cerevisiae.

We have constructed a non-primate lentiviral vector system based on the equine infectious anaemia virus (EIAV). This system is able to transduce both dividing and non-dividing cells, including primary cultured hippocampal neurons and neurons and glia in the adult rat central nervous system (CNS), at efficiencies comparable with HIV-based vectors. We demonstrate that the only EIAV proteins required for this activity are gag/pol and that the only accessory protein required for vector production is rev. In addition, we show that the pol encoded dUTPase activity that is found in all non-primate lentiviruses is not required. The vectors can be pseudotyped with a range of envelopes including rabies G and MLV 4070A and can be concentrated to high titres. The ability of EIAV to infect mitotically inactive cells makes this vector an attractive alternative to the immunodeficiency viruses for gene therapy.

Stable gene transfer to the nervous system using a non-primate lentiviral vector

The use of human immunodeficiency virus vectors for gene therapy is hampered by concern over their safety. This concern might be ameliorated, in part, if the viral accessory genes and proteins could be eliminated from the vector genomes and particles. Here we describe a minimal vector system that is capable of transducing nondividing cells and which does not contain tat, vif, vpr, vpu, and nef.

Minimal Requirement for a Lentivirus Vector Based on Human Immunodeficiency Virus Type 1

The 5' control region of the yeast phosphoglycerate kinase gene (PGK) was fused to the coding sequence of a human interferon-alpha. This PGK-interferon fusion was then introduced into yeast on a high copy number 2mu-based plasmid vector. Strains containing this plasmid produced a PGK-interferon-alpha fusion protein as 1-2% of cell protein and the expression of interferon activity was regulated by the availability of a fermentable carbon source. The system is capable of making as much as 15 mg of human interferon-alpha per litre of batch culture.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1982.tb01215.x

A J Kingsman

Papers

Conservation of high efficiency promoter sequences in Saccharomyces cerevisiae.

Stable gene transfer to the nervous system using a non-primate lentiviral vector

Minimal Requirement for a Lentivirus Vector Based on Human Immunodeficiency Virus Type 1

Regulated high efficiency expression of human interferon-alpha in Saccharomyces cerevisiae.

HIV-1 TAT “activates” presynthesized RNA in the nucleus