Hybrid MR-PET of brain tumours using amino acid PET and chemical exchange saturation transfer MRI.

PURPOSE: PET using radiolabelled amino acids has become a promising tool in the diagnostics of gliomas and brain metastasis. Current research is focused on the evaluation of amide proton transfer (APT) chemical exchange saturation transfer (CEST) MR imaging for brain tumour imaging. In this hybrid MR-PET study, brain tumours were compared using 3D data derived from APT-CEST MRI and amino acid PET using O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET). METHODS: Eight patients with gliomas were investigated simultaneously with 18F-FET PET and APT-CEST MRI using a 3-T MR-BrainPET scanner. CEST imaging was based on a steady-state approach using a B1 average power of 1µT. B0 field inhomogeneities were corrected a Prametric images of magnetisation transfer ratio asymmetry (MTRasym) and differences to the extrapolated semi-solid magnetisation transfer reference method, APT# and nuclear Overhauser effect (NOE#), were calculated. Statistical analysis of the tumour-to-brain ratio of the CEST data was performed against PET data using the non-parametric Wilcoxon test. RESULTS: A tumour-to-brain ratio derived from APT# and 18F-FET presented no significant differences, and no correlation was found between APT# and 18F-FET PET data. The distance between local hot spot APT# and 18F-FET were different (average 20 ± 13 mm, range 4-45 mm). CONCLUSION: For the first time, CEST images were compared with 18F-FET in a simultaneous MR-PET measurement. Imaging findings derived from18F-FET PET and APT CEST MRI seem to provide different biological information. The validation of these imaging findings by histological confirmation is necessary, ideally using stereotactic biopsy.

Use of visual CO2 feedback as a retrofit solution for improving classroom air quality.

Carbon dioxide (CO2 ) sensors that provide a visual indication were installed in classrooms during normal school operation. During 2-week periods, teachers and students were instructed to open the windows in response to the visual CO2 feedback in 1 week and open them, as they would normally do, without visual feedback, in the other week. In the heating season, two pairs of classrooms were monitored, one pair naturally and the other pair mechanically ventilated. In the cooling season, two pairs of naturally ventilated classrooms were monitored, one pair with split cooling in operation and the other pair with no cooling. Classrooms were matched by grade. Providing visual CO2 feedback reduced CO2 levels, as more windows were opened in this condition. This increased energy use for heating and reduced the cooling requirement in summertime. Split cooling reduced the frequency of window opening only when no visual CO2 feedback was present.

Mestizos with systemic lupus erythematosus develop renal disease early while antimalarials retard its appearance: data from a Latin American cohort.

OBJECTIVES: The objective of this paper is to assess the predictors of time-to-lupus renal disease in Latin American patients. METHODS: Systemic lupus erythematosus (SLE) patients (n = 1480) from Grupo Latino Americano De Estudio de Lupus (GLADEL's) longitudinal inception cohort were studied. Endpoint was ACR renal criterion development after SLE diagnosis (prevalent cases excluded). Renal disease predictors were examined by univariable and multivariable Cox proportional hazards regression analyses. Antimalarials were considered time dependent in alternative analyses. RESULTS: Of the entire cohort, 265 patients (17.9%) developed renal disease after entering the cohort. Of them, 88 (33.2%) developed persistent proteinuria, 44 (16.6%) cellular casts and 133 (50.2%) both; 233 patients (87.9%) were women; mean (± SD) age at diagnosis was 28.0 (11.9) years; 12.2% were African-Latin Americans, 42.5% Mestizos, and 45.3% Caucasians (p = 0.0016). Mestizo ethnicity (HR 1.61, 95% CI 1.19-2.17), hypertension (HR 3.99, 95% CI 3.02-5.26) and SLEDAI at diagnosis (HR 1.04, 95% CI 1.01-1.06) were associated with a shorter time-to-renal disease occurrence; antimalarial use (HR 0.57, 95% CI 0.43-0.77), older age at onset (HR 0.90, 95% CI 0.85-0.95, for every five years) and photosensitivity (HR 0.74, 95% CI 0.56-0.98) were associated with a longer time. Alternative model results were consistent with the antimalarial protective effect (HR 0.70, 95% CI 0.50-0.99). CONCLUSIONS: Our data strongly support the fact that Mestizo patients are at increased risk of developing renal disease early while antimalarials seem to delay the appearance of this SLE manifestation. These data have important implications for the treatment of these patients regardless of their geographic location.

Cerebral amyloid angiopathy and microhemorrhages after amyloid beta vaccination: case report and brief review.

After the interruption of the international multicenter Phase 2 clinical trial with active immunotherapy based on synthetic Abeta42 (AN1792), few reports about the neuropathological findings in those patients and those from the Phase 1 clinical trial were published. These reports described some pathological similarities among the patients such as a reduction in the burden of amyloid plaques, the reactions of microglia/macrophages and the persistence of neurofibrillary tangles and neuropil threads. In addition, a lymphocytic inflammatory infiltrate as well as white matter lesions were present in some cases with meningoencephalitis. In both animal models of vaccination, as well as some vaccinated patient samples, there appears to be a correlation between vaccination and hemorrhages. Subsequently, two series reports concerning 8 patients from the Phase 1 initial trial showed that immunization with Abeta42 seemed to result in clearance of amyloid plaques, but did not prevent progressive neurodegeneration and that it increased vessel wall amyloid angiopathy as well as cortical microhemorrhages. Recent clinical data gave further encouraging results regarding vaccination in humans demonstrating that long term follow-up of patients from the second clinical trial revealed reduced functional decline, at least, in antibody responders. Here we describe a patient diagnosed with Alzheimer's disease who also participated in the Phase 2 clinical trial. A neuropathological examination confirmed Alzheimer's disease without meningoencephalitis and revealed a severe amyloid angiopathy with frequent microhemorrhages, the decrease of amyloid load being difficult to ascertain. Our results are in agreement with previous studies and confirm the presence of severe amyloid angiopathy after vaccination. The latter may be a transient phenomenon depending on the degree of immune response and the pathological stage of the disease when the patient underwent treatment. In addition, our vaccinated case also demonstrated microhemorrhages and microinfarcts which were already noticed to occur with a higher density in immunized Alzheimer's disease patients.

Acute rheumatic fever. Still a challenge.

At the end of the 20th century, after an apparent decline, acute rheumatic fever (ARF) now constitutes a great challenge for developed and developing countries. It is caused by a group A beta-hemolytic Streptococcus upper airways infection, but the exact pathogenetic mechanisms are not yet clear. The role of the immune system in the pathogenesis of ARF is understood better than genetic host factors. ARF can mimic many other diseases, and the diagnosis is based on clinical criteria. It is still overdiagnosed and underdiagnosed in different settings. Penicillin has greatly contributed to the reduction in the incidence and recurrence of this disease. Current schemes of prophylaxis, however, present many problems, and failures are common. Future efforts to reduce the burden of this disease should induce public health measures the vaccine strategies.

Studies of host-plasmid interactions in recombinant microorganisms.

Plasmid genes redirect some components of cellular metabolism into synthesis of plasmid gene products and additional plasmids. The stoichiometric and kinetic implications of these host-plasmid interactions have been investigated theoretically and experimentally. Using known pathway energetics, maximum theoretical yield factors based on ATP, glucose, and O2 have been estimated for recombinant Escherichia coli and compared with corresponding estimates for host cells alone, indicating major changes in carbon and energetic stoichiometry in recombinant cells in cases of high cloned gene expression. The influence of the number of plasmids in recombinant E. coli has been experimentally characterized using a series of pMB1 derivatives stably propagated at copy numbers from 12 to 408. Recombinant cell growth rate declines monotonically as plasmid content increases as does efficiency of plasmid gene expression. A detailed metabolically structured single-cell model for E. coli has successfully simulated these trends. Interrelationships among number of plasmids per cell, induction of expression of a plasmid gene, and recombinant population growth rate have been experimentally delineated for Saccharomyces cerevisiae containing plasmid pLGSD5 and derivatives in which the 2-micron origin of replication has been replaced by a cloned ARS1 sequence or its deletion fragments. The CEN4 centromere sequence has been included in some of these plasmids to provide more regular segregation. Specific growth rate of these recombinant yeasts exhibits a maximum as a function of plasmid content, an effect attributed to the interplay between beneficial effects of the plasmid in selective medium and parasitic effects on metabolism at larger plasmid content or with more plasmid gene expression activity. The yeast strains investigated exhibit substantial segregational instability that was characterized using a rapid-flow cytometry measurement based upon single-cell deletion of E. coli beta-galactosidase activity in recombinant cells.

Catabolite repression and induction time effects for a temperature-sensitive GAL-regulated yeast expression system.

The effects of residual catabolite repression and the importance of induction timing were determined for a temperature-sensitive (ts) GAL-regulated stable yeast expression system. The Saccharomyces cerevisiae strain employed carries a reg1 mutation inhibiting catabolite repression, and a ts mutation enabling induction of the regulated GAL promoters by a temperature shift to 35 degrees C. Despite the reg1 mutation and induction method, glucose depressed lacZ expression from a GAL1 promoter during batch culture. beta-Galactosidase specific activity was consistently lower at higher initial glucose concentrations in both SDC (semi-defined) and YPDa (complex) media; decreases of 18-36% were observed as glucose concentration was increased between 1, 3, 5, and 10 g l-1. However, the reductions in beta-galactosidase specific activity due to residual catabolite repression were more than balanced by substantial improvements in biomass yield at higher glucose levels. Therefore, productivity rose with increasing glucose concentration; in YPDa medium, increasing initial glucose from 1 to 10 g l-1 resulted in a 2.6-fold increase in beta-galactosidase volumetric activity. Due to the negative effects of shifting temperature to 35 degrees C, the trade-offs between optimum growth and a lengthy induction period were also evaluated. Delaying the time of induction reduced final specific activities but improved cell yield, and waiting 14 h into batch culture to induce lacZ expression provided modest 9-15% improvements in overall productivity.

Estimation of the rate of cloned gene integration via retrotransposition.

A theoretical method has been developed to estimate retrotransposition (integration) rates of the Saccharomyces cerevisiae Ty3 and Ty1 retrotransposons bearing heterologous and homologous genes (neor, HIS3). The method is based on population growth modeling and Lea and Coulson's maximal likelihood method for mutation rate estimation (Lea and Coulson, 1949). This method has allowed us to examine directly retrotransposition rates of GAL-regulated marked Ty3 and Ty1 elements into the whole yeast genome, not just a particular DNA sequence, for the purpose of cloned gene integration. The integration rates of a Ty3-neor system (ca. 1 x 10(-3) cell-1 generation-1) and a Ty1-neor system (ca. (2-3) x 10(-3) cell-1 generation-1) were not significantly affected by temperature (18 and 30 degrees C). However, the retrotransposition rate of the Ty3-neor-HIS3 system increased from ca. 2 x 10(-5) to 2 x 10(-4) cell-1 generation-1 when the temperature was decreased from 30 to 18 degrees C. The retrotransposition rate of Ty3-neor was significantly higher than that of Ty3-neor-HIS3 and slightly lower than that of Ty1-neor. This method can be used to estimate integration rates of other Ty3 and Ty1 elements and to evaluate the efficiency of Ty-mediated cloned gene integration.

Enhanced productivity through gratuitous induction in recombinant yeast fermentations.

The advantages of gratuitous induction for GAL-regulated cloned gene (lacZ) product synthesis were evaluated for the yeast Saccharomyces cerevisiae. The growth, yield, and productivity of a gratuitous (gal1) strain were compared with those of an otherwise isogenic, nongratuitous (GAL1) strain. Batch studies clearly demonstrated the improvements possible in product synthesis when the inducer is not metabolized by the yeast cells; both beta-galactosidase specific and volumetric activities were superior for the gal1 strain. At equivalent metabolizable sugar concentrations, the productivity of the gratuitous strain exceeded that of the nongratuitous strain by 180%. The effects of initial inducer concentration and induction time were also examined. For the gratuitous strain, galactose:glucose ratios as low as 0.1 still gave maximum beta-galactosidase volumetric activity. A 5-fold higher ratio was necessary for full induction with the nongratuitous strain, and productivity was substantially lower relative to the gal1 strain. A comparison of various times for galactose addition indicated that productivity is highest when the gratuitous culture is induced for the entire batch fermentation.

Ty1-mediated integration of expression cassettes: host strain effects, stability, and product synthesis.

The yeast retrotransposon Ty1 has been used to insert multiple copies of heterologous genes into the genome of Saccharomyces cerevisiae. Amplification using a GAL1-regulated Ty1 element carrying a 4.6 kilobase pair expression cassette (Escherichia coli lacZ structural gene under the control of the yeast CUP1 promoter, and the bacterial neo gene) was compared with that of a GAL1-regulated Ty1 element carrying only the neo gene. Mobilization of Ty1 was induced from a chromosomal element and a 2 mu-plasmid-based element; similar results were obtained for both locations. The two marked Ty1 cassettes were successfully integrated into the genomes of three different S. cerevisiae strains. Efficiencies were found to vary significantly between strains. The size of the inserted cassette was also important; the efficiency for the CUP1p-lacZ-neo cassette was much lower than that for the neo cassette in the same host. All amplified copies were found to be quite stable with or without expression for at least 50 generation in nonselective medium; moreover, there were no significant effects on the growth of the cells. After integration, beta-galactosidase specific activity from the lacZ construct in the three hosts was found to correlate well with the copy number of the CUP1p-lacZ expression cassette amplified by the Ty1 retrotransposon.

Sequential delta-integration for the regulated insertion of cloned genes in Saccharomyces cerevisiae.

A novel delta-integration vector was developed to allow the sequential insertion of multiple cloned genes in the yeast Saccharomyces cerevisiae. To allow repetitive integrations, the reusable URA3 Blaster selection cassette was employed; the insertions (of CUP1p-lacZ in this study) were selected using the URA3 marker which was subsequently "popped" out by recombination between flanking direct repeats. Transformants contained only one new integrated copy after the loss of the URA3 marker, and subsequent transformations were effective for the sequential insertion of a series of genes (one at a time) into dispersed chromosomal delta sequences. The structural stability of the integrations was location-dependent (ranging from 75% to 100% after 50 generations in complex medium with or without gene expression), and the integrations (at least up to five) had no significant effects on the growth of the cells. In addition, beta-galactosidase specific activity levels varied linearly with integrated copy number. The repetitive, regulated nature of integration with this vector is not possible with traditional delta-integration or other homologous recombination methods, and is promising for fine-tuning cloned gene copy number and for the insertion of metabolic pathway genes.

Unruptured intracranial aneurysm as a cause of cerebral ischemia.

BACKGROUND: Unruptured intracranial artery aneurysms (IAs) can be revealed by cerebral ischemia. Little is known on the clinical course and outcome of patients with this condition. We report our findings in a consecutive series of 15 such patients. METHODS: We retrospectively analyzed patients with ischemic stroke (IS) or transient ischemic attack (TIA), unruptured IA on the symptomatic cerebral artery, and no other potential cause of cerebral ischemia consecutively treated in a tertiary stroke unit. RESULTS: Fifteen patients (ten women, and five men) were identified. Their mean age was 49.7 years (range, 37-80 years). Ten patients presented with IS, and five with TIA. The median diameter of IA was 7.5mm (range, 2.5-23 mm). Aneurysm thrombosis was found on imaging in 9/10 patient with IS, and 1/5 patients with TIA (p=0.017). Thirteen patients were given an antiplatelet agent. Mean follow-up until last visit or treatment of aneurysm was 393 days (median 182 days; range, 6-1825 days). There was no ischemic recurrence. Partial or complete recanalization of aneurysm thrombosis occurred in 7/10 patients. Two patients, both with initial aneurysmal thrombosis and on antiplatelet therapy, experienced aneurysm rupture. CONCLUSION: Unruptured IA is a rare cause of IS/TIA. IS is associated with aneurysm thrombosis. Our findings suggest that aneurysm thrombosis is a dynamic process which is associated with a low rate of ischemic recurrence on antiplatelet therapy but may be followed by subarachnoid hemorrhage.

Improved protein synthesis and secretion through medium enrichment in a stable recombinant yeast strain.

Two Saccharomyces cerevisiae strains were employed to investigate the effects of medium enrichment on the expression and secretion of a recombinant protein. One was a stable autoselection strain with mutations in the ura3, fur1, and urid-k genes. The combination of these three mutations blocks both the pyrimidine nucleotide biosynthetic and salvage pathways and is lethal to the cells. Retention of the plasmid, which carries a URA3 gene, was essential for cell viability. Therefore, all media were selective, allowing cultivation of the strain in complex medium. The second strain was a nonautoselection (control) strain and is isogenic to the first except for the fur1 and urid-k mutations. The plasmid utilized contains the yeast invertase gene under the control of the MFalpha1 promoter and leader sequence. The expression and secretion of invertase for the autoselection strain were examined in batch culture for three media: a minimal medium (SD), a semidefined medium (SDC), and a rich complex medium (YPD). Biomass yields and invertase productivity (volumetric activity) increased with the complexity of the medium; total invertase volumetric activity in YPD was 100% higher than in SDC and 180% higher than in SD. Specific activity, however, was lowest in the SDC medium. Secretion efficiency was extremely high in all three media; for the majority of the culture, 80-90% of the invertase was secreted into the periplasmic space and/or culture medium. A glucose pulse at the end of batch culture in YPD facilitated the transport of residual cytoplasmic invertase. For the nonautoselection strain, invertase productivity did not improve as the medium was enriched from SDC to YPD, and plasmid stability in the complex YPD medium dropped from 54% to 34% during one batch fermentation. During long-term sequential batch culture in YPD, invertase activity decreased by 90% and the plasmid-containing fraction dropped from 56% to 8.8% over 44 generations of growth. The expression level for the autoselection strain, however, remained high and constant over this time period, and no reversion at the fur1 or urid-k locus was observed.

Evaluation of pKD1-based plasmid systems for heterologous protein production in Kluyveromyces lactis.

The stability of pKD1-based vectors was evaluated during the synthesis of intracellular and extracellular gene products in the yeast Kluyveromyces lactis. The Escherichia coli lacZ and MFalpha1 leader-BPTI (bovine pancreatic trypsin inhibitor) cassettes were placed under the control of the inducible K. lactis LAC4 promoter and inserted into the pKD1-based plasmids. To induce gene expression while maintaining inducer level, a gratuitous gal1-209 K. lactis strain was employed. Selective medium containing 5 g glucose/l and 0.5 g galactose (inducer)/l allowed optimum expression and secretion of heterologous products without a significant effect on the growth of the recombinant cells. During long-term sequential batch cultures (60 generations), plasmid instability was mainly the result of structural instability. The expression and secretion of BPTI resulted in greater structural instability relative to the intracellular beta-galactosidase. For both products, vectors carrying the pKD1 replication origin and the cis-acting stability locus (partial-pKD1 vectors) were more stable than vectors carrying the full pKD1 sequence (full-pKD1 vectors). However, after 55 generations, the beta-galactosidase and BPTI activities were still higher with the full-pKD1 vectors. This was due to the significantly higher initial beta-galactosidase and BPTI activities for the full-pKD1 vectors (approximately 85% and 47% higher, respectively) relative to the partial-pKDI vectors. Southern blots confirmed that these increases were due to the higher copy number of the vectors carrying the full pKD1 sequence. In contrast to our previously reported results for the secretion of invertase, full-pKD1 vectors were preferred for the expression/secretion of beta-galactosidase and BPTI for at least 55 generations. Due to their structural stability, partial-pKD1 vectors will be advantageous for very long cultivation times.

Site-specific integration of heterologous genes in yeast via Ty3 retrotransposition.

The Ty3 retrotransposon of Saccharomyces cerevisiae was employed for the site-specific integration of heterologous genes into the yeast genome. A GAL-regulated promoter allowed induction of the retrotransposition process, and a bacterial neo(r) gene inserted in the Ty3 element was used as a selectable model heterologous gene. The frequency of transposition of this neo(r)-marked element was found to be comparable to that of an unmarked element. Three amplification systems were constructed; the systems varied with respect to the location and number of the GAL-regulated helper and neo(r)-marked Ty3 elements. For all three systems, neo(r) integrations were readily selected with a maximum of two insertions obtained per round of amplification. A sequential amplification strategy was effective for further increasing the number of integrated cloned genes, and families of strains varying by only one neo(r) insertion were easily obtained. Resistance to the antibiotic G418 correlated well with the number of integrated neo(r) genes, and Northern blots verified the relationship between cloned gene number (up to four) and neo(r) expression. Structural stability of the integrated genes was also demonstrated. By controlling the number of rounds of amplification and the level of G418 selection, precise numbers of integrated heterologous genes could be obtained. Because the amplification process can be repeated using different cloned genes inserted in the Ty3 element, these results demonstrate the potential of retrotransposition for the regulated integration of a series of different genes at nondeleterious chromosomal locations.

Enhancement of cloned gene product synthesis via autoselection in recombinant Saccharomyces cerevisiae.

Saccharomyces cerevisiae autoselection strains with mutations in the ura3, fur1, and urid-k genes have been obtained through a sequential isolation procedure. This autoselection system is an extension of one described by Loison et al. The mutations effectively block both the pyrimidine biosynthetic and salvage pathways and in combination are lethal to the host. Therefore, a plasmidencoded URA3 gene is essential for cell viability regardless of the growth conditions, and complex (traditionally nonselective) media can be employed without the risk of plasmid loss. The effects of medium enrichment on growth and cloned gene product synthesis were examined in batch culture for two autoselection strains. The plasmid gene product beta-galactosidase was under the control of the yeast GAL1 promoter, and two methods of induction were employed; one strain was induced via temperature shift while the other was induced by galactose addition. Three nutrient media were investigated: a lean selective medium (SD), a richer semidefined medium (SDC), and a rich complex medium (YPD). The results demonstrated the improvements in cloned gene productivity possible when the growth medium is enriched, with up to 10-fold increases in beta-galactosidase productivity observed. Plasmid instability and mutation reversion were not problems for the autoselection strains, even in uracil-containing medium. Short-term plasmid stabilities were approximately 90% in all three media tested. During continuous culture of the autoselection temperature-sensitive strain, long-term plasmid stability was excellent and beta-galactosidase expression remained high after more than 25 residence times under inducing conditions. In contrast, both beta-galactosidase specific activity and plasmid stability decreased linearly with time for an analogous nonautoselection strain. The introduced fur1 and uridk mutations were very stable; after more than 50 generations of growth in complex medium, stability values of 99-100% were measured.

Influence of dilution rate and induction of cloned gene expression in continuous fermentations of recombinant yeast.

The effects of growth rate on cloned gene product synthesis in recombinant Saccharomyces cerevisiae have been studied in continuous culture. The plasmid employed contains a yeast GAL10-CYC1 hybrid promoter directing expression of the E. coli lacZ gene. beta-Galactosidase production was therefore controlled by the yeast galactose regulatory circuit, and the induction process and its effects were studied at the various dilution rates. At all dilution rates plasmid stability decreased with induction of lacZ gene expression. In some instances, two induced "steady states" were observed, the first 10-15 residence times after induction and the second after 40-50 residence times. The second induced steady state was characterized by greater biomass concentration and lower beta-galactosidase specific activity relative to the first induced "steady-state." beta-Galactosidase specific activity and biomass concentration increased as dilution rate was reduced, and despite lower flow rate and plasmid stability, overall productivity (activity/L/hr) was substantially higher at low dilution rate. Important factors influencing all of the trends were the glucose and galactose (inducer) concentrations in the vessel and inducer metabolism.

Influence of plasmid origin and promoter strength in fermentations of recombinant yeast.

The effects of plasmid promoter strength and origin of replication on cloned gene expression in recombinant Saccharomyces cerevisiae have been studied in batch and continuous culture. The plasmids employed contain the Escherichia coli lacZ gene under the control of yeast promoters regulated by the galactose regulatory circuit. The synthesis of beta-galactosidase was therefore induced by the addition of galactose. The initial induction transients in batch culture were compared for strains containing plasmids with 2mu and ARS1 origins. As expected, cloned gene product synthesis was much lower with the ARS1 plasmid: average beta-galactosidase specific activity was an order of magnitude below that with the 2mu-based plasmid. This was primarily due to the low plasmid stability of 7.5% when the plasmid origin of replication was the ARS1 element. The influence of plasmid promoter strength was studied using the yeast GAL1, GAL10, and hybrid GAL10-CYC1 promoters. The rate of increase in beta-galactosidase specific activity after induction in batch culture was 3-5 times higher with the GAL1 promoter. Growth rate under induced conditions, however, was 15% lower than in the absence of lacZ expression for this promoter system. The influence of plasmid promoter strength on induction behavior and cloned gene expression was also studied in continuous fermentations. Higher beta-galactosidase production and lower biomass concentration and plasmid stability were observed for the strain bearing the plasmid with the stronger GAL1 promoter. Despite the decrease in biomass concentration and plasmid stability, overall productivity in continuous culture using the GAL1 promoter was three times that obtained with the GAL10-CYC1 promoter.

G418 Selection and stability of cloned genes integrated at chromosomal delta sequences of Saccharomyces cerevisiae.

The chromosomal delta sequences of the yeast Saccharomyces cerevisiae were employed as recombination sites to integrate the bacterial neo(r) gene and the yeast SUC2 gene into the yeast genome. A dominate selection method employing the aminoglycoside antibiotic G418 was used. Transformation efficiencies and growth behaviors of the transformants were studied. Transformants were obtained with more than 40 integrations; the majority of insertions were tandem with a maximum of three different insertion sites utilized at one time. After 70-100 generations of growth in nonselective medium, the high copy number SUC2-neo(r) integrants were found to be unstable; only minor instability was observed for the neo(r) and low copy number SUC2-neo(r) integrants.

Development of a LAC4 promoter-based gratuitous induction system in Kluyveromyces lactis.

A gratuitous induction system based on the strong, indigenous LAC4 promoter was developed for Kluyveromyces lactis. To prevent consumption of the inducer galactose, a strain with a gal1-209 mutation was employed; this mutation disables the galactokinase function but retains the regulatory function for induction. The Escherichia coli lacZ gene (encoding beta-galactosidase) is functional in K. lactis and was used as the reporter gene downstream of the LAC4 promoter on a multicopy plasmid. The gal1-209 strain exhibited several unexpected phenomena, including partial consumption of the inducer galactose (although at a much slower rate relative to GAL1 strains) and growth inhibition at high concentrations of galactose. These unusual characteristics, however, did not prevent the successful construction of a strong gratuitous induction system. Due to the low rate of inducer consumption for the gratuitous strain, very low concentrations of galactose (1:20 galactose:glucose) resulted in high-level induction. Under these conditions, beta-galactosidase specific and volumetric activities were 4.2- and 5.5-fold higher, respectively, than those for the "GAL1" nongratuitous strain. This research demonstrated the improved productivity possible via LAC4 promoter-based gratuitous induction (and thus a more stable inducer concentration). The effects of various carbon source concentrations on growth and induction were also determined.