We developed a methylotrophic yeast, Pichia pastoris, as a host for DNA transformations. The system is based on an auxotrophic mutant host of P. pastoris which is defective in histidinol dehydrogenase. As a selectable marker, we isolated and characterized the P. pastoris HIS4 gene. Plasmid vectors which contained either the P. pastoris or the Saccharomyces cerevisiae HIS4 gene transformed the P. pastoris mutant host. DNA transfer was accomplished by a modified version of the spheroplast generation (CaCI2-polyethylene glycol)-fusion procedure developed for S. cerevisiae. In addition, we report the isolation and characterization of P.pastoris DNA fragments with autonomous replication sequence activity. Two fragments, PARS1 and PARS2, when present on plasmids increased transformation frequencies to 105/,ug and maintained the plasmids as autonomous elements in P. pastonis cells.
Pim-1 kinase is a member of a distinct class of serine/ threonine kinases consisting of Pim-1, Pim-2, and Pim-3. Pim kinases are highly homologous to one another and share a unique consensus hinge region sequence, ER-PXPX, with its two proline residues separated by a nonconserved residue, but they (Pim kinases) have <30% sequence identity with other kinases. Pim-1 has been implicated in both cytokine-induced signal transduction and the development of lymphoid malignancies. We have determined the crystal structures of apo Pim-1 kinase and its AMP-PNP (5-adenylyl-,␥-imidodiphosphate) complex to 2.1-Å resolutions. The structures reveal the following. 1) The kinase adopts a constitutively active conformation, and extensive hydrophobic and hydrogen bond interactions between the activation loop and the catalytic loop might be the structural basis for maintaining such a conformation. 2) The hinge region has a novel architecture and hydrogen-bonding pattern, which not only expand the ATP pocket but also serve to establish unambiguously the alignment of the Pim-1 hinge region with that of other kinases. 3) The binding mode of AMP-PNP to Pim-1 kinase is unique and does not involve a critical hinge region hydrogen bond interaction. Analysis of the reported Pim-1 kinase-domain structures leads to a hypothesis as to how Pim kinase activity might be regulated in vivo.
A target nucleic acid sequence can be replicated (amplified) exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H, and a DNAdependent RNA polymerase. By mimickisg the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target. Product accumulation is exponential with respect to time, indicating that newly synthesized cDNAs and RNAs function as templates for a continuous series of transcription and reverse transcription reactions. Ten million-fold amplification occurs after a 1-to 2-hr incubation, with an initial rate of amplification of 10-fold every 2.5 min. This self-sustained sequence replication system is useful for the detection and nucleotide sequence analysis of rare RNAs and DNAs. The analogy to aspects of retroviral replication is discussed.The transfer of genetic information from RNA to DNA and then back to RNA is a scheme characteristic of retroviruses. Such a scheme provides a mechanism for the replication of RNA genomes (reviewed in refs. 1-3). In exploring variations of an in vitro transcription-based amplification system (TAS) (4), it was discovered that it was possible to devise a concerted, three-enzyme, in vitro reaction to carry out an isothermal replication of target nucleic acid sequences, analogous to the strategy used in retroviral replication. This reaction is a self-sustained sequence replication (3SR) system involving the collective activities of avian myeloblastosis virus (AMV) reverse transcriptase, Escherichia coli RNase H, and T7 RNA polymerase. The accumulation ofboth target nucleic acid-specific RNA Approximately 25 mg (wet weight) of Trisacryl beads containing oligonucleotide 86-273 (5'-AGTCTAGCAGAA-GAAGAGGTAGTAATTAGA-3') was prehybridized in 250 pA of hybridization solution (5 x standard saline phosphate/ EDTA/10% dextran sulfate/0.1% SDS) in a 2-ml microcolumn (Isolab) for 30 min at 370C. The prehybridization solution was removed, and 40 p.1 of fresh hybridization solution was added to the beads, together with the 60 p.1 of solution from the solution hybridization step. The beads were then incubated at 370C for 1 hr with occasional mixing and then washed six times with 1 ml of 2x standard saline citrate (6) at 370C. The radioactivity of the beads and the combined washes was measured by Cerenkov counting for 1 min. The amount of target detected was determined by calculating the percentage of total radioactivity captured on the beads and Abbreviations: 3SR, self-sustained sequence replication; TAS, transcription-based amplification system; HIV-1, human immunodeficiency virus type 1; AMV, avian myeloblastosis virus.§To whom reprint requests should be addressed. 1874The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Proc. Natl. Acad. Sci...
Drug susceptibility and mutations in the reverse transcriptase (RT) gene were analyzed with 167 virus isolates from 38 patients treated with nevirapine, a potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) RT. Resistant isolates emerged quickly and uniformly in all patients administered nevirapine either as monotherapy or in combination with zidovudine (AZT). Resistance developed as early as 1 week, indicating rapid turnover of the virus population. The development of resistance was associated with the loss of antiviral drug activity as measured by CD4 lymphocyte counts and levels of HIV p24 antigen and RNA in serum. In addition to mutations at amino acid residues 103, 106, and 181 that had been identified by selection in cell culture, mutations at residues 108, 188, and 190 were also found in the patient isolates. Sequences from patient clones documented cocirculating mixtures of populations of different mutants. The most common mutation with monotherapy, tyrosine to cysteine at residue 181, was prevented from emerging by coadministration of AZT, which resulted in the selection of alternative mutations. The observations documented that, under selective drug pressure, the circulating virus population can change rapidly, and many alternative mutants can emerge, often in complex mixtures. The addition of a second RT inhibitor, AZT, significantly altered the pattern of mutations in the circulating population of HIV.
In Pichia pastoris, alcohol oxidase (AOX) is the first enzyme in the methanol utilization pathway and is encoded by two genes, AOXI and AOX2. The DNA and predicted amino acid sequences of the protein-coding portions of the genes are closely homologous, whereas flanking sequences share no homology. The functional roles ofAOXI and AOX2 in the metabolism of methanol were examined. Studies of strains with disrupted AOX genes revealed that AOXI was the major source of methanol-oxidizing activity in methanol-grown P. pastoris. The results of two types of experiments each suggested that the difference in AOX activity contributed by the two genes was a consequence of sequences located 5' of the protein-coding portions of the genes. First, the coding portion of AOX2 was able to functionally substitute for that of AOXI when placed under the control of AOXI regulatory sequences. Second, when labeled oligonucleotide probes specific for the 5' nontranslated region of each gene were used, it was apparent that the steady-state level ofAOXI mRNA was much higher than that ofAOX2. Except for the difference in the amount of mRNA present, the two genes appeared to be regulated in the same manner. A physiological reason for the existence of AOX2 was sought but was not apparent.A key feature which distinguishes methanol-metabolizing yeasts from bacteria is the reaction mechanism used to oxidize methanol to formaldehyde (2,46,47). Whereas a dehydrogenase linked to the electron transport chain catalyzes this reaction in bacteria, yeasts and fungi have an oxygen-dependent oxidase as the electron acceptor. In addition to methanol, this enzyme oxidizes other lower primary aliphatic alcohols: hence the name alcohol oxidase (AOX; EC 1.1.3.13). The enzyme functions as an octamer of identical subunits, each of which contains one noncovalently bound flavin adenine dinucleotide moiety. AOX has a low affinity for oxygen, and cells compensate for this poor catalytic ability by synthesizing large amounts of the enzyme. In methylotrophic yeasts, AOX is not detectable in glucose-grown cells, but makes up as much as 30% of the total soluble protein in methanol-grown cells (8). In two methylotrophic yeasts, Pichia pastoris and Hansenula polymorpha, synthesis of AOX is known to be controlled at the transcriptional level (20,27,34). Because the oxidation of methanol generates hydrogen peroxide, AOX is sequestered in a subcellular organelle termed the microbody or peroxisome (46,47). Unlike the transport of proteins to many organelles, such as the mitochondria, endoplasmic reticulum, and vacuoles (18,23,38), the compartmentalization of AOX and most other peroxisomal proteins does not involve a processed amino-terminal signal sequence (21,26). In peroxisomes, octamers of AOX form into and apparently function as a crystalloid structure, giving these large peroxisomes a striking cube-shaped appearance within the yeast (46,47).The isolation and partial characterization of one AOX gene, AOXI, from Pichia pastoris has been described (20). Fusion vector studies...
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