Structural basis for calcium and phosphatidylserine regulation of phospholipase C δ1

Many membrane-associated enzymes, including those of the phospholipase C (PLC) superfamily, are regulated by specific interactions with lipids. Previously, we have shown that the C2 domain of PLC δ1 is required for phosphatidylserine (PS)-dependent enzyme activation and that activation requires the presence of Ca(2+). To identify the site of interaction and the role of Ca(2+) in the activation mechanism, we mutagenized three highly conserved Ca(2+) binding residues (Asp-653, Asp-706, and Asp-708) to Gly in the C2 domain of PLC δ1. The PS-dependent Ca(2+) binding affinities of the mutant enzymes D653G, D706G, and D708G were reduced by 1 order of magnitude, and the maximal level of Ca(2+) binding was reduced to half of that of the native enzyme. The level of Ca(2+)-dependent PS binding was also reduced in the mutant enzymes. Under basal conditions, the Ca(2+) dependence and the maximal level of hydrolysis of phosphatidylinositol 4,5-bisphosphate were not altered in the mutants. However, the Ca(2+)-dependent PS stimulation was severely defective. PS reduces the K(m) of the native enzyme almost 20-fold, but far less for the mutants. Replacing Asp-653, Asp-706, and Asp-708 simultaneously with glycine in the C2 domain of PLC δ1 leads to a complete and selective loss of the stimulation and binding by PS. These results show that D653, D706, and D708 are required for Ca(2+) binding in the C2 domain and demonstrate a mechanism by which C2 domains can mediate regulation of enzyme activity by specific lipid ligands.     

Rad53 is essential for a mitochondrial DNA inheritance checkpoint regulating G1 to S progression

The Chk2-mediated deoxyribonucleic acid (DNA) damage checkpoint pathway is important for mitochondrial DNA (mtDNA) maintenance. We show in this paper that mtDNA itself affects cell cycle progression. Saccharomyces cerevisiae rho(0) cells, which lack mtDNA, were defective in G1- to S-phase progression. Deletion of subunit Va of cytochrome c oxidase, inhibition of F(1)F(0) adenosine triphosphatase, or replacement of all mtDNA-encoded genes with noncoding DNA did not affect G1- to S-phase progression. Thus, the cell cycle progression defect in rho(0) cells is caused by loss of DNA within mitochondria and not loss of respiratory activity or mtDNA-encoded genes. Rad53p, the yeast Chk2 homologue, was required for inhibition of G1- to S-phase progression in rho(0) cells. Pif1p, a DNA helicase and Rad53p target, underwent Rad53p-dependent phosphorylation in rho(0) cells. Thus, loss of mtDNA activated an established checkpoint kinase that inhibited G1- to S-phase progression. These findings support the existence of a Rad53p-regulated checkpoint that regulates G1- to S-phase progression in response to loss of mtDNA.     

Ethanol production from henequen (Agave fourcroydes Lem.) juice and molasses by a mixture of two yeasts

In the fermentation process of henequen (Agave fourcroydes Lem.) leaf juice, complemented with industrial molasses, the use of an inoculum comprising two yeasts: Kluyveromyces marxianus (isolated from the henequen plant) and Saccharomyces cerevisiae (commercial strain) was studied. An ethanol production of 5.22+/-1.087% v/v was obtained. Contrary to expected, a decrease on ethanol production was observed with the use of the K. marxianus strain. The best results were obtained when a mixture of 25% K. marxianus and 75% S. cerevisiae or S. cerevisiae alone were used with an initial inoculum concentration of 3x10(7)cellmL(-1). Furthermore, it was possible to detect a final concentration of approximately 2-4gL(-1) of reducing sugars that are not metabolized by the yeasts for the ethanol production. These results show that although the use of a mixture of yeasts can be of interest for the production of alcoholic beverages, it can be the opposite in the case of ethanol production for industrial purposes where manipulation of two strains can raise the production costs.     

Involvement of Rho-type GTPase in control of cell size in Saccharomyces cerevisiae

Maintaining specific cell size, which is important for many organisms, is achieved by coordinating cell growth and cell division. In the budding yeast Saccharomyces cerevisiae, the existence of two cell-size checkpoints is proposed: at the first checkpoint, cell size is monitored before budding at the G1/S transition, and at the second checkpoint, actin depolymerization occurring in the small bud is monitored before the G2/M transition. Morphological analyses have revealed that the small GTPase Rho1p participates in cell-size control at both the G1/S and the G2/M boundaries. One group of rho1 mutants (rho1A) underwent premature entry into mitosis, leading to the birth of abnormally small cells. In another group of rho1 mutants (rho1B), the mother cells failed to reach an appropriate size before budding, and expression of the G1 cyclin Cln2p began at an earlier phase of the cell cycle. Analyses of mutants defective in Rho1p effector proteins indicate that Skn7p, Fks1p and Mpk1p are involved in cell-size control. Thus, Rho1p and its downstream regulatory pathways are involved in controlling cell size in S. cerevisiae.     

Control of Lactobacillus contaminants in continuous fuel ethanol fermentations by constant or pulsed addition of penicillin G

The addition of penicillin G to combat microbial contamination in continuous fuel alcohol fermentations was performed using both continuous and pulsed addition regimes. In continuous fermentations where both Saccharomyces cerevisiae and Lactobacillus paracasei were present, the mode of addition of penicillin G determined final numbers of viable L. paracasei. When the same overall average concentration of penicillin G was added in both pulsed and continuous modes, the initial viable number of L. paracasei (8.0 x 10(9) cfu ml(-1)) decreased to a greater degree (1.02 x 10(5) cfu ml(-1) L. paracasei) when penicillin G was pulsed at 6 h frequencies at an overall average concentration of 2,475 U/l than when penicillin G was added continuously at 2,475 U/l (2.77 x 10(5) cfu ml(-1) L. paracasei). Pulsed additions over longer frequencies at 2,475 U/l were not as effective in reducing viable bacteria. Viable yeasts increased during both treatment conditions by more than 2-fold. The two addition regimes also eliminated the 40% decrease in ethanol concentration caused by the intentional bacterial infection. Although there was 3 times more bacterial death with 6 h pulsed additions compared to continuous additions of penicillin G at 2,475 U/l, there was, by that point, no practical difference in either final ethanol concentration or relative ethanol recovery.     

The respiratory burst response to Histoplasma capsulatum by human neutrophils. Evidence for intracellular trapping of superoxide anion

Human neutrophils (PMN) have received little attention as to the role they play in host defense against Histoplasma capsulatum (Hc). We have characterized the binding and phagocytosis of Hc yeasts by human PMN and quantified the PMN respiratory burst in response to this organism. mAb specific for CD11a, CD11b, and CD11c all partially blocked the attachment of unopsonized yeasts to PMN; a mAb to CD18 inhibited attachment by greater than 90%. Thus, human PMN recognize and bind Hc yeasts via CD18 adhesion receptors as has been found for human cultured macrophages and alveolar macrophages. Unopsonized yeasts were phagocytosed by PMN, but phagocytosis was increased markedly by heat-labile and heat-stable serum opsonins. These opsonins promoted enhanced phagocytosis of yeasts by increasing the attachment of Hc yeasts to the PMN membrane. Phagocytosis of viable or heat-killed Hc yeasts by PMN did not induce the secretion of superoxide anion (O2-) as quantified by the reduction of cytochrome c. O2- was not detected when yeasts were opsonized in normal serum or immune serum, or at a ratio of yeasts to PMN of up to a 100:1. However, phagocytosis of opsonized yeasts by PMN did not prevent them from subsequently releasing O2- after further incubation with opsonized zymosan or PMA. Opsonized Hc yeasts clearly stimulated the PMN respiratory burst as quantified by intracellular reduction of nitroblue tetrazolium, reduction of cytochrome c in the presence of cytochalasin D, oxygen consumption, luminol-enhanced and nonenhanced chemiluminescence, and H2O2 production. These data suggest that phagocytosis of Hc yeasts by PMN is associated with intracellular entrapment of O2- that is not detectable by reduction of extracellular cytochrome c.     

Electrostatic domino effect in the Shaker K channel turret

Voltage-gated K channels are regulated by extracellular divalent cations such as Mg(2+) and Sr(2+), either by screening of fixed negative surface charges, by binding directly or close to the voltage sensor, or by binding to the pore. Different K channels display different sensitivity to divalent cations. For instance, 20 mM MgCl(2) shifts the conductance versus voltage curve, G(V), of the Kv1-type Shaker channel with 14 mV, while the G(V) of Kv2.1 is shifted only with 7 mV. This shift difference is paralleled with different working ranges. Kv1-type channels open at approximately -20 mV and Kv2.1 channel open at approximately +5 mV. The aim of this study was to identify critical residues for this Mg(2+)-induced G(V) shift by introducing Kv2.1 channel residues in the Shaker K channel. The K channels were expressed in Xenopus laevis oocytes and studied with the two-electrode voltage-clamp technique. We found that three neutral-to-positive amino-acid residue exchanges in the extracellular loops connecting transmembrane segments S5 and S6 transferred the Mg(2+)-shifting properties. The contributions of the three residues were additive, and thus independent of each other, with the contributions in the order 425 > 419 > 451. Charging 425 and 419 not only affect the Mg(2+)-induced G(V) shift with 5-6 mV, but also shifts the G(V) with 17 mV. Thus, a few strategically placed surface charges clearly modulate the channel's working range. Residue 425, located at some distance away from the voltage sensor, was shown to electrostatically affect residue K427, which in turn affects the voltage sensor S4-thus, an electrostatic domino effect.     

Use of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae to modify the lipid composition and function of mitochondrial membranes

KD115 (ol1), an unsaturated fatty acid auxotroph of S. cerevisiae, was grown in a semi-synthetic medium supplemented with 3.3 x 10(-4) M palmitoleic (cis 16:1) or palmitelaidic (trans 16:1) acids. The parent strain S288C was studied as a control. The lipid composition (fatty acids, neutral lipids, and phospholipids), respiratory activity (O2 consumption), and ultrastructure were compared in mutant yeast grown with each unsaturated fatty acid supplement. The fatty acid supplement represented 70-80% of the yeast fatty acids. Yeast grown in trans 16:1 contained more squalene, a higher ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC), and had 10-20% of the respiratory activity compared to the same yeast grown in cis 16:1. The mitochondrial morphology of yeast in each growth supplement was notably different. The use of mixtures of cis and trans 16:1 in different proportions revealed that the PE/PC ratio, the squalene content, the respiratory defect, and the mitochondrial morphology were all similarly dependent on the fraction of trans 16:1 in the mixtures. As little as 10-20% of cis 16:1 in the mixture was sufficient to abrogate the physiological effects of trans 16:1 on each of the parameters noted above. The combined effects of high content of trans unsaturated fatty acid and the altered phospholipid composition seem to account for the decrease in lipid fluidity, the defective structure and function of the mitochondrial membrane.     

Steady-state and transient-state analyses of aerobic fermentation in Saccharomyces kluyveri

Some yeasts, such as Saccharomyces cerevisiae, produce ethanol at fully aerobic conditions, whereas other yeasts, such as Kluyveromyces lactis, do not. In this study we investigated the occurrence of aerobic alcoholic fermentation in the petite-negative yeast Saccharomyces kluyveri that is only distantly related to S. cerevisiae. In aerobic glucose-limited continuous cultures of S. kluyveri, two growth regimens were observed: at dilution rates below 0.5 h(-1) the metabolism was purely respiratory, and at dilution rates above 0.5 h(-1) the metabolism was respiro-fermentative. The dilution rate at which the switch in metabolism occurred, i.e. the critical dilution rate, was 66% higher than the typical critical dilution rate of S. cerevisiae. The maximum specific oxygen consumption rate around the critical dilution rate was found to 13.6 mmol (g dry weight)(-1) h(-1) and the capacity of the pyruvate dehydrogenase-bypass pathway was estimated to be high from in vitro enzyme activities; especially the specific activity of acetyl-CoA synthetase was much higher than in S. cerevisiae at all tested conditions. Addition of glucose to respiring cells of S. kluyveri led to ethanol formation after a delay of 20-50 min (depending on culture conditions prior to the pulse), which is in contrast to S. cerevisiae that ferments immediately after glucose addition.     

Modeling the Current-Voltage Characteristics of Charophyte Membranes. II. The Effect of Salinity on Membranes of Lamprothamnium papulosum

Lamprothamnium is a salt-tolerant charophyte that inhabits a broad range of saline environments. The electrical characteristics of Lamprothamnium cell membranes were modeled in environments of different salinity: full seawater (SW), 0.5 SW, 0.4 SW, and 0.2 SW. The cells were voltage-clamped to obtain the I/V (current-voltage) and G/V (conductance-voltage) profiles of the cell membranes. Cells growing at the different salinities exhibited one of three types of I/V profiles (states): pump-, background- and K(+)-states. This study concentrates on the pump- and background-states. Curved (pump-dominated) I/V characteristics were found in cells with resting membrane PDs (potential differences) of -219 +/- 12 mV (in 0.2 SW: 6 cells, 16 profiles), -161 +/- 12 mV (in 0.4 SW: 6 cells, 7 profiles), -151 +/- 12 mV (in 0.5 SW: 6 cells, 12 profiles) and -137 +/- 12 mV (in full SW: 8 cells, 13 profiles). The linear I/V characteristics of the background-state were found in cells with resting PDs of -107 +/- 12 mV (in 0.4 SW: 7 cells, 12 profiles), -108 +/- 12 mV (in 0.5 SW: 7 cells, 10 profiles) and -104 +/- 12 mV (in full SW: 3 cells, 5 profiles). The resting conductance (G) of the cells progressively increased with salinity, from 0.5 S x m(-2) (in 0.2 SW) to 22.0 S x m(-2) (in full SW). The pump peak conductance only rose from 2 S x m(-2) (0.2 SW) to 5 S x m(-2) (full SW), accounting for the increasingly depolarized resting PD observed in cells in more saline media. Upon exposure to hypertonic medium, both the pump and an inward K+ rectifier were stimulated. The modeling of the I/V profiles identified the inward K+ rectifier as an early electrical response to hypertonic challenge.     

酿酒酵母呼吸缺陷型和野生型酒精发酵特性的比较分析*

比较了酒精发酵生产菌株IFFI1300及其呼吸缺陷型突变株在酒精产量、发酵动力学、耐酒精能力及与酒精发酵相关的乙醇脱氢酶活性等方面的特性。结果表明：1)发酵终期的酒精产量,45株呼吸缺陷型的平均值与野生型没有显著性差异;但部分缺陷型的酒精产量高于野生型。2)酒精发酵动力学结果显示,呼吸缺陷型酒精产生速度略高于野生型。3)单位重量干菌体的乙醇脱氢酶活性,呼吸缺陷型高于野生型。以上结果提示：呼吸缺陷型用于酒精发酵以提高酒精产量和缩短发酵周期是有潜力的。4)单位体积发酵液的乙醇脱氢酶活性则野生型高于呼吸缺陷型,主要原因在于呼吸缺陷型的生物量明显低于野生型。5)呼吸缺陷型菌株之间的耐酒精能力差别很小,耐酒精能力的高低与酒精产量的高低没有明显的正相关性。一般的,酒精产量高的菌株耐酒精能力较强。在实验结果的基础上,对呼吸缺陷型用于酒精发酵的优越性和可行性进行了讨论。     

Structure of the O-antigen polysaccharide present in the lipopolysaccharide of Cronobacter dublinensis (subspecies lactaridi or lausannensis) HPB 3169

Cronobacter dublinensis (formerly Enterobacter sakazakii) HPB 3169 is a pathogenic Gram-negative bacterium that produces a smooth-type lipopolysaccharide in which the antigenic O-polysaccharide component was determined to be a repeating pentasaccharide unit composed of L-rhamnose; 2-acetamido-2-deoxy-D-glucose; 3,6-dideoxy-3-(R)-3-hydroxybutyramido-D-glucose; and 3-deoxy-manno-oct-2-ulosonic acid in the respective molar ratio 2:1:1:1. Chemical and 2D NMR analyses of the O-polysaccharide and a pentasaccharide derived by the mild acid hydrolysis of the ketosyl linkage of the Kdo (3-deoxy-D-manno-2-octulosonic acid) residue in the O-polysaccharide established that the O-antigen is a high molecular mass unbranched polymer of a repeating pentasaccharide unit and has the structure [see formula in text] where Bu is a (R)-3-hydroxybutanoyl substituent. The O-antigen is structurally similar to that of the recently reported Cronobacter sakazakii strain G706 (designated as serotype O5), except that in strain G706 the d-Qui3N is in its N-acetyl form, in contrast to its presence as a 3-deoxy-3-(R)-3-hydroxybutyramido derivative in the C. sakazakii HPB 3169 strain O-antigen.     

Estrogen metabolism as measured in blood and urine in female rhesus monkeys

In order to measure the interconversions of estrone (E1) and estradiol (E2) and their conversion to the 16 alpha-hydroxylated estrogens, 16 alpha-hydroxy estrone (16 alpha-OHE1) and estriol (E3), we infused 11 female rhesus monkeys with [3H]E2 and [14C]E1 and measured radioactivity in the blood as E1, E2 and 16 alpha-OHE1 (n = 9) and in the urine as the glucuronides of E1, E2, 16 alpha-OHE1, and E3 (n = 11). The mean conversion of E1 to E2 as measured in blood (percent of infused E1 measured in blood as E2, [rho]1.2BB) was 29.2 +/- 1.6% and as measured in the urine of the same animals, [rho]1.2BM, was 77.4 +/- 5.9%. The mean conversion of E2 to E1, [rho]2.1BB was 21.5 +/- 1.0% and as measured in urine, [rho]2.1BM was 67.7 +/- 4.6%. Thus for both estrone and estradiol only 30-35% of the interconversions occurred in pools which were in equilibrium with the blood pool of these estrogens. The remaining 65-70% occurred in a pool, probably liver, in which glucuronidation occurred immediately after conversion. The conversion ratios (the ratio of the concentration in the blood of radioactivity as 16 alpha-OHE1 to its precursor, CRPrec,16 alpha-OHE1) was 0.036 +/- 0.008 for CRE1,16 alpha-OHE1 and 0.0039 +/- 0.0010 for CRE2,16 alpha-OHE1. The percentages of administered E1 excreted in the urine as the glucuronides of E1, E2, 16 alpha-OHE1 and E3 were 20.1 +/- 1.5, 1.6 +/- 0.2, 0.96 +/- 0.20 and 0.76 +/- 0.07 respectively. The percentages of administered E2 excreted in the urine as E1, E2, 16 alpha-OHE1 and E3 were 14.4 +/- 1.0, 2.2 +/- 0.3, 0.57 +/- 0.05 and 0.68 +/- 0.11 respectively. Thus there are minor differences in the patterns of excreted metabolites of E1 and E2. Furthermore, 16 alpha-OHE1 and E3 are not major metabolites of E1 or E2 in the female rhesus monkey.     

Cell-cycle-specific activators of the Mec1/ATR checkpoint kinase

Mec1 [ATR (ataxia telangiectasia mutated- and Rad3-related) in humans] is the principle kinase responsible for checkpoint activation in response to replication stress and DNA damage in Saccharomyces cerevisiae. The heterotrimeric checkpoint clamp, 9-1-1 (checkpoint clamp of Rad9, Rad1 and Hus1 in humans and Ddc1, Rad17 and Mec3 in S. cerevisiae; Ddc1-Mec3-Rad17) and the DNA replication initiation factor Dpb11 (human TopBP1) are the two known activators of Mec1. The 9-1-1 clamp functions in checkpoint activation in G1- and G2-phase, but its employment differs between these two phases of the cell cycle. The Ddc1 (human Rad9) subunit of the clamp directly activates Mec1 in G1-phase, an activity identified only in S. cerevisiae so far. However, in G2-phase, the 9-1-1 clamp activates the checkpoint by two mechanisms. One mechanism includes direct activation of Mec1 by the unstructured C-terminal tail of Ddc1. The second mech-anism involves the recruitment of Dpb11 by the phosphorylated C-terminal tail of Ddc1. The latter mechanism is highly conserved and also functions in response to replication stress in higher eukaryotes. In S. cerevisiae, however, both the 9-1-1 clamp and the Dpb11 are partially redundant for checkpoint activation in response to replication stress, suggesting the existence of additional activators of Mec1.     

Use of whey protein beads as a new carrier system for recombinant yeasts in human digestive tract

A new immobilizing protocol using whey protein isolates was developed to entrap recombinant Saccharomyces cerevisiae. The model yeast strain expresses the heterologous P45073A1 that converts trans-cinnamic acid into p-coumaric acid. Beads resulted from a cold-induced gelation of a whey protein solution (10%) containing yeasts (7.5 x 10(7)cells ml(-1)) into 0.1M CaCl(2). The viability and growth capability of yeasts were not altered by our entrapment process. The release and activity of immobilized yeasts were studied in simulated human gastric conditions. During the first 60 min of digestion, 2.2+/-0.9% (n=3) of initial entrapped yeasts were recovered in the gastric medium suggesting that beads should cross the gastric barrier in human. The P45073A1 activity of entrapped yeasts remained significantly higher (p<0.05) than that of free ones throughout digestion (trans-cinnamic acid conversion rate of 63.4+/-1.6% versus 51.5+/-1.8% (n=3) at 120 min). The protein matrix seemed to create a microenvironment favoring the activity of yeasts in the stringent gastric conditions. These results open up new opportunities for the development of drug delivery system using recombinant yeasts entrapped in whey protein beads. The main potential medical applications include biodetoxication or the correction of digestive enzyme deficiencies.     

Detection of Escherichia coli O157:H7 using immunomagnetic separation and absorbance measurement

An assay system for detection of Escherichia coli O157:H7 was developed based on immunomagnetic separation of the target pathogen from samples and absorbance measurement of p-nitrophenol at 400 nm from p-nitrophenyl phosphate hydrolysis by alkaline phosphatase (EC 3.1.3.1) on the "sandwich" structure complexes (antibodies coated onto micromagnetic beads--E. coli O157:H7-antibodies conjugated with the enzyme) formed on the microbead surface. The effects of immunoreaction time, phosphate buffer concentration, pH and temperature on the immunomagnetic separation of E. coli O157:H7 from samples were determined and the conditions used for the separation were 1-h reaction time, 1.0 x 10(-2) M PBS, pH 8.0 and 33 degrees C in this system. The effects of MgCl(2) concentration, Tris buffer concentration, pH and temperature on the activity of alkaline phosphatase conjugated on the immuno-"sandwich" structure complexes were investigated after immunomagnetic separation of the target pathogen and the conditions used for the enzymatic amplification were 1.0 x 10(-4) M MgCl(2), 1.0 M Tris buffer, pH 8.0, 28 degrees C and 30-min reaction time during the assay. The selectivity of the system was examined and no interference from the other pathogens including Salmonella typhimurium, Campylobacter jejuni and Listeria monocytogenes was observed. Its working range was from 3.2 x 10(2) to 3.2 x 10(4) CFU/ml, and the relative standard deviation was 2.5-9.9%. The total detection time was less than 2 h.     

三种食用植物酵素中酵母菌的分离鉴定及耐受性试验

从自然发酵的火龙果酵素、青梅酵素和葡萄酵素中筛选酵母菌,对筛选到的菌株进行鉴定及耐受性试验。从两种火龙果酵素中筛选到3株菌:P_1、P_2和P_3,从青梅酵素中筛选到2株菌:PM_1和PM_2,从葡萄酵素中筛选到2株菌:G_1和G_2;分子生物学鉴定结果显示:P_3、PM_1、PM_2和G_2为鲁氏接合酵母,P_1为酿酒酵母,P_2和G_1为丘陵假丝酵母。耐受性试验结果表明:筛选出的菌株在起始pH为2. 5、3. 0、3. 5以及初始葡萄糖含量为300、400、600、750 g/L的培养基上均可生长,其中P_2、PM_1和G_1在培养基起始pH为2. 0时可生长;P_2在培养基起始pH为1. 5时可生长;筛选出的菌株均具有耐低pH和耐高糖的特性。     

The A1 x U72 base pair conserved in eukaryotic initiator tRNAs is important specifically for binding to the eukaryotic translation initiation factor eIF2.

The formation of a specific ternary complex between eukaryotic initiation factor 2 (eIF2), the initiator methionyl-tRNA (Met-tRNA), and GTP is a critical step in translation initiation in the cytoplasmic protein-synthesizing system of eukaryotes. We show that the A1 x U72 base pair conserved at the end of the acceptor stem in eukaryotic and archaebacterial initiator methionine tRNAs plays an important role in this interaction. We changed the A1 x U72 base pair of the human initiator tRNA to G1 x C72 and expressed the wild-type and mutant tRNA genes in the yeast Saccharomyces cerevisiae by using constructs previously developed in our laboratory for expression of the human initiator tRNA gene in yeasts. We show that both the wild-type and mutant human initiator tRNAs are aminoacylated well in vivo. We have isolated the wild-type and mutant human initiator tRNAs in substantially pure form, free of the yeast initiator tRNA, and have analyzed their properties in vitro. The G1 x C72 mutation affects specifically the binding affinity of eIF2 for the initiator tRNA. It has no effect on the subsequent formation of 40S or 80S ribosome initiator Met-tRNA-AUG initiation complexes in vitro or on the puromycin reactivity of the Met-tRNA in the 80S initiation complex.