You will say that it might have been the voice of an Asiatic — of an African. Neither Asiatics nor Africans abound in Paris; but, without{l} denying the inference, I will{m} now merely call your attention to three points.{n} The voice is termed by one witness ‘harsh rather than shrill.’ It is represented by two others to have been ‘quick and unequal.’ No words — no sounds{o} resembling words — were{p} by any witness mentioned as distinguishable.

whisker

agility

Jardin des Plantes

“I am now awaiting,” continued he, looking toward{z} the door of our apartment — “I am now awaiting a person who, although perhaps not the perpetrator of these butcheries, must have been{a} in some measure implicated in their perpetration. Of the worst portion of the crimes committed, it is probable that he is innocent.

“The Purloined Letter,”

I do not mean to say they are not ingenious — but people think they are more ingenious than they are — on account of their method and air of method

Because adding research to your essays requires the use of others’ writing, and because many assignment require you to write about others’ writing, it is possible to plagiarize accidentally through incorrect citation.

Using ideas from elsewhere requires correct citation, and using ideas from elsewhere without citation is plagiarism.

sensuous lust

a courtesan-girl, a hetaera

gazelles

tyrannical

deities

she-asses

brass

impart

kons-9 is that it combines the power of a software development IDE with the visual tools of 3D graphics authoring system. It does this by being implemented in Common Lisp, an object-oriented dynamic language which provides powerful facilities for exploratory development and rapid prototyping within a live interactive software environment

REPL-based

I figure that most of the learning students lost in Zoom school is learning they would have lost by early adulthood even if schools had remained open.

Most grown-ups believe that school is only for learning back-to-back. They don’t understand the parts that build character for us teens.

2.7-9 Theorem (Continuity and boundedness)

2.6-9 Theorem (Range and null space).

rocessing of special categories of personal data 45. Subject to compliance with the Data Protection Regulation and any other relevant enactment or rule of law, the processing of special categories of personal data shall be lawful to the extent the processing is— (a) authorised by section 41 and sections 46 to 54 , or (b) otherwise authorised by Article 9.

collecting and checking the content of declarations of private interests, of personal data that are liable to disclose indirectly the political opinions, trade union membership or sexual orientation of a natural person constitutes processing of special categories of personal data, for the purpose of those provisions.

those provisions cannot be interpreted as meaning that the processing of personal data that are liable indirectly to reveal sensitive information concerning a natural person is excluded from the strengthened protection regime prescribed by those provisions, if the effectiveness of that regime and the protection of the fundamental rights and freedoms of natural persons that it is intended to ensure are not to be compromised.

ZFIN: ZDB-ALT-160119-9

surveillance capitalism, invented by Google in 2001, benefitted from a couple of important historical windfalls. One is that it arose in the era of a neoliberal consensus around the superiority of self-regulating companies and markets. State-imposed regulation was considered a drag on free enterprise. A second historical windfall is that surveillance capitalism was invented in 2001, the year of 9/11. In the days leading up to that tragedy, there were new legislative initiatives being discussed in Congress around privacy, some of which might well have outlawed practices that became routine operations of surveillance capitalism. Just hours after the World Trade Center towers were hit, the conversation in Washington changed from a concern about privacy to a preoccupation with “total information awareness.” In this new environment, the intelligence agencies and other powerful forces in Washington and other Western governments were more disposed to incubate and nurture the surveillance capabilities coming out of the commercial sector.

User participation in any online internet community generally follows the 90-9-1 rule:90% of community members are lurkers who read or observe, but don’t contribute9% of community members edit or respond to content but don’t create content of their own1% of community members create new content

in which we retaliated for an attack on our soil

These designers value expression over style

syntactical

Shattering the constraints of minimalism was exhilarating and far more fun than the antiseptic discipline of the classical Swiss school.

ssrp1as819

Roberts

As a result, the Texas law is a permissible regulation of speech.

Only certain religious groups are free to participate.

Stevens: dissenting

vote of 7–2

schools and programs

Separate Opinions

Clark: concurring in the judgment of the Court

Happiness

For example, saver's credits make certain retirement savings tax-exempt, meaning savers get to keep more of the interest they earn on savings investments.

In the United States and many other countries, the government taxes gains from private investment. Low capital gains taxes encourage investment and so also economic growth.

RRID:ZFIN_ZDB-ALT-180517-9

Results for individual PALB2 variants were normalized relative to WT-PALB2 and the p.Tyr551ter (p.Y551X) truncating variant on a 1:5 scale with the fold change in GFP-positive cells for WT set at 5.0 and fold change GFP-positive cells for p.Y551X set at 1.0. The p.L24S (c.71T>C), p.L35P (c.104T>C), p.I944N (c.2831T>A), and p.L1070P (c.3209T>C) variants and all protein-truncating frame-shift and deletion variants tested were deficient in HDR activity, with normalized fold change <2.0 (approximately 40% activity) (Fig. 1a).

A total of 84 PALB2 patient-derived missense variants reported in ClinVar, COSMIC, and the PALB2 LOVD database were selected

SUPPLEMENTARY DATA

To this end, 44 missense variants found in breast cancer patients were identified in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar) and/or selected by literature curation based on their frequency of description or amino acid substitution position in the protein (Supplemental Table S1).

Source Data

Source Data

We, therefore, analyzed the effect of 48 PALB2 VUS (Fig. 2a, blue) and one synthetic missense variant (p.A1025R) (Fig. 2a, purple)29 on PALB2 function in HR.

Most Suspected Brugada Syndrome Variants Had (Partial) Loss of Function

we selected 73 previously unstudied variants: 63 suspected Brugada syndrome variants and 10 suspected benign variants

He was so deeply offended and concerned by the notion that somehow it was America’s fault that a group of radical, violent Islamist terrorists killed nearly 3,000 Americans that it opened his eyes to the indoctrination that was happening in his classes.

Supplemental material

Supplemental material

We analysed a total of 82 blood samples derived from 77 individuals (online supplemental table 3). These 77 individuals corresponded either to new index cases suspected to harbour a pathogenic TP53 variant or to relatives of index cases harbouring TP53 variants.

select the transfer option in Intello

Figure 9 shows the decreasing optical transmit-tance at the visible region with increasingfilm thickness(increasing immersion time)

Men who worship me,thinking solely of me,always disciplined,win the reward I secure.

I am the universal father,mother, granter of all, grandfather,object of knowledge, purifier,holy syllable OM, threefold sacred lore.

article D. 111-9 du code de l’éducation

71 1. The Standard Version, Tablet IX [He] clad himself in their skins, he ate their flesh. Gilgamesh [dug] wells that never existed before, [he] drank the water, as he chased the winds. Sham ash grew worried, and bending down, he spoke to Gilgamesh: '0 Gilgamesh, where are you wandering? The life that you seek you never will find.' Said Gilgamesh to him, to the hero Shamash: 'After roaming, wandering all through the wild, when I enter the Netherworld will rest be scarce? I shall lie there sleeping all down the years! 'Let my eyes see the sun and be sated with light! The darkness is hidden, how much light is there left? When may the dead see the rays of the sun?'

The life that you seek you never will find.'

Christian Reclamation Center

a drive-in restaurant

THE BUDDHIST church up on the hill next to a playground

Überwachung

Überwachungs

Acute toxicity studies

TLC study of phenols

Analysis of internal transcribed spacer region

Effect of temperature on xylanase activity

Effect of pH on xylanase activity

Effect of various carbon sourceson xylanase activity

Assayof xylanase activity

Dinitrosalicylate reagent (DNS)(per liter)

Citrate phosphate buffer

Reagents

Xylanase activity

Harvesting of cultures

Enzyme production (EP) medium

Inoculum preparation

Sporulation medium used for Trichodermasp

Maintenance of Trichodermasp. culture

Sterilization

Materials for xylanase induction

Induction of xylanase from Trichodermasp

Detection of hemolytic activity

Statistical analysis

Urease production

Urease productio

Plasmid curing of Vibriofrom Cochin estuary,shrimp farms and seafood

In silico analysis of sequenced Dof domain and Dof genes

Assay for reduced glutathione(GSH)

Assay for Lipid peroxidase(LPO)

Estimation of Total protein

Oxidative stress parameters

Quantification of ROS

Detection of intracellular ROS

Acridine orange/ethidium bromide (AO –EB) staining

Cytomorphological analysis

MTT [(4, 5–dimethylthiazol–2–yl)–2, 5-diphenyl tetrazolium Bromide] assay

Cell culture

Micronuclei test

SDS -PAGE for serum protein analysis

Estimation of serum proteins (SDS –PAGE) and Micronuclei in fish, Labeo rohita

HarvestIndex

Cell surface and Intracellular staining and Flow Cytometric Analysis

Low-pH antibody elution buffer

Luciferase assay in SW480 cells under nickel induced condition

pET30-b-APC Nde/Sca (1211-1495 a.a) (antigen for APC antibody)

Transformation ofStm754 with Ea-cmyc-CiST plasmid

IFN Biossay

Expression of costimulatory molecules on the vaccines

Phenotypic analysis by Flow cytometry

Long PCR

Polymerase Chain Reaction (PCR)

ImmunoprecipitationofVEGF

Endomorphic mesomorph:

Biceps

Purified HbS was digested with carboxypeptidase B (200 mg hemoglobin to 1 mg of the enzyme) for 3 hours in freshly prepared 0.05 M Tris-acetate buffer pH 7.1) at 25°C , followed by passage through a cation-exchange column using Whatman CM52.

Preparation of HbS{des arg 141a

Protein concentrations were detennined using BCA protein estimation kit (Pierce, .. USA). The assay was perfonned according to the instructions provided by the manufacturer. Various dilutions of the sample or BSA were made in appropriate buffer and 200 J.ll of supplied reagent mix (1 :50 ratio) was added to each well in a 96 well plate. The plate was incubated at 37°C for 1 h and the absorbance was measured at 540 nrn.

Protein Estimation

antibody at an appropriate dilution. The immunoreactivity was detected by enhanced chemiluminescence using an ECL detection kit (Amersham Biosciences) and were recorded on X-ray films after appropriate exposure and development. It is important to note that the blots for probing phosphorylated proteins were performed using 1% BSA as blocking agent instead ofblotto

Whole cell extracts were prepared by treating cells with lysis buffer (0.125M Tris, 4% SDS, 20% glycerol, and 10% ~-mercaptoethanol), and protein estimation was performed using CB-X protein assay kit as per manufacturer's protocol. Lysates were resolved on 12% SDS-PAGE gel, following which Western transfer was performed onto nitrocellular membranes using a BioRad Western transfer apparatus. The blots were incubated with 5% blotto (non-fat dry skimmed milk) in 0.05% PBS-Tween 20 for 1 h to block non-specific binding sites following which they were incubated for 1 h with primary antibody at an appropriate dilution prepared in 1% blotto in 0.05% PBS-Tween-20. The blots were washed 3x with 0.05% PBS-Tween-20 at 5 min intervals following which they were incubated for 1 h with secondary

SDS-PAGE and Western blot

Sequencing of cloned inserts was done by Sanger's dideoxy chain termination method (Sanger et al., J 977) using Sequenase version 2.0 kit from USB. 5 J.ll of J mg/ml suspension of plasmid DNA was incubated in denaturation buffer at 37 °C for 30 min. in a reaction volume of 50 J.ll, and then the precipitation was carried out in the presence of 5.5 J.ll of 3M sodium acetate and 4 volumes of chilled ethanol at -70 °C for 30 min. The pellet, obtained by centrifugation at 10,000 g, for 20 min., at 4 °C, was washed with 70% ethanol and resuspended in 7 J.ll of sterile water. J pmole of sequencing primer in J J.ll water and 2J.ll of 5X sequenase reaction buffer were added to denatured DNA and the reaction mix was incubated at 65 °C for 5 min. for primer annealing. The reaction mixture was cooled slowly to about 35 °C, by putting the heat block at room temperature. For labeling, J J.ll of 0. J M DTT, J J.ll radioactivity containinig J 0 J.lCi of 35S dATP, 2 Jlllabeling mix diluted 5-fold in steriJe water and 2 Jll sequenase enzyme diluted 8-fold in sequenase dilution buffer were added to the· primer annealed DNA. Incubated the reaction mixture at room temperature for 2-5 min. and added 3.5 J.ll to each of the 4 different tubes containing 2.5 J.ll dideoxy nucleotides ddATP, ddTTP, ddCTP, and ddGTP separately. The mixture was incubated at 37 °C for 5 min. and finally, the reaction was stopped by adding 4 J.ll of estop solution to each tube. Reaction products were separated on a 6% polyacrylamide sequencing gel made in TBE buffer containing 7.5 M urea. The samples were heated at 75 °C for 2 min. and immediately loaded on the gel. The gel was run at a constant power of 60 watts maintaining the temperature of gel between 50-55 °C, dried and exposed to an X-ray film.

DNA Sequencing

r-bZP3 was arsanilated using a modification of the procedure of Nisnoff (1967). Briefly, arsanilic acid (100 mg) was dissolved in 5 ml of I M HCI. A IO ml stock of NaN02 (10 mg/ml) was also prepared fresh and added dropwise to the arsanilic acid solution while vortexing. Activation of arsanilic acid was checked on starch-KI paper. The ice cold activated arsanilic acid solution was added dropwise to the protein solution (5 mg of r-bZP3 in 100 mM PB, pH 7.4) stirring constantly in an ice water bath, while pH was maintained between 9.0 and 9.5 with 10 N NaOH. The protein solution was dialyzed extensively against I 00 mM PB having 4 M urea. Arsanilation of r-bZP3 was checked by ELISA using ars-r-bZP3 for coating ( 1 flg/well) and using a 1:100 dilution of murine anti-ars MAb, R 16.7 (Durdik et al., 1 989). Bound Ab was revealed using anti-mouse HRPO conjugate (I :5000). Three monkeys previously immunized with r-bZP3-DT conjugate (MRA-375, MRA-640 and MRA-672) and 2 naive monkeys (MRA-446 and MRA-670) were immunized at 2 intramuscular sites with 250 flg of ars-bZP3 conjugate using Squalene:Arlacel A ( 4: 1) as an adjuvant. Boosters were administered at intervals of 20 days and bleeds were collected I 0 days post immunization. Bleeds were analyzed by ELISA using r-bZP3 and ars-BSA for coating to determine anti-bZP3 and anti-ars Ab titres as described earlier.

Arsanilation of r-bZP3

Cells and supernatant collected 72 h pi were analyzed on a 0.1% SDS-I 0% PAGE and Western blot using polyclonal Abs generated in rabbit against peptide-DT conjugates using (i) 23-45 aa residue N-terminal peptide with an extra lysine at the N-terminus (KQPFWLLQGGASRAETSVQPVLVE), (ii) 300-322 aa residue C-terminal peptide (CSFSKSSNSWFPVEGPADICQCC) corresponding to the bZP3 sequence (iii) MA-451 and (iv) goat anti-GST Ab. The C-terminal anti-peptide Ab was used for determining whether or not the full length bZP3 was being expressed by the cells infected with the different viruses. Anti-mouse (I :500), anti-rabbit ( 1 :500) or anti-goat ( 1 :500) Abs conjugated to HRPO were used for revealing bound Ab.

Analysis ofr-bZP3 in VI, V2, V3 and V4 Infected Cells and Supernatant

From an 0/N grown culture, 1 ml cells were pelleted in a 1.5 rnl eppendorf tube. The cells wer~ washed once with 100 ul of solution I ( 50 rnM glucose in 25 rnM Tris. HCl ,· pH 8. 0 ) . The cells were pelleted again and resuspended in 70 ul of solution I. To this, 20 ul of a freshly prepared solution of lysozyme 10 rng 1 ml in distilled water was added. The tube was vortexed to mix the contents and incubated in ice for 5 minutes. Next, 10 ul of 0.1 M EDTA, pH 8.0, was added, vortexed and the tube incubated in ice for 5 minutes. Next, 200 ul of solution IV ( 0.2 N NaOH + 1 % SDS was added, the contents vortexed quickly but briefly to mix and incubated in ice for 5 minutes. Finally, 150 ul of 5 M potassium acetate, pH 4. 8 was added and the tube incubated in ice. After 60 minutes, the tube was centrifuged for 10 minutes at 10,000 rpm, at 4°C. 450 ul of the supernate was removed to another tube and DNA precipitated with two volumes of ethanol at -7 0°C for 15 minutes. The DNA pellet was collected by centrifugation and after draining off the supernate, the pellet was washed with 80 % ethanol. The pellet was dried briefly under vacuum and finally resuspended in 150 ul TE. From this, a 10 ul aliquot was used for checking on gel or for•setting up digestions with restriction endon~cleases.

Plasmid DNA minipreps.

Nitrocellulose membranes ( BA85 ) were obtained from Schleicher and Schuell, Germany. GeneScreen and GeneScreen Plus membranes were from DuPont, USA. Millipore membranes ( 0.45 um ) were from Millipore Corporation, USA. 3 MM and 1 MM chromatography filter papers were from Whatman Ltd, U.K.

other Materials.

Selected transformants were grown in 250 ml of LBamp 0/N. The cells from the 0/N cultures were harvested by centrifugation (4°C) at 4000 X g for 30 min. The cell pellet was resuspended in 5 ml of TEG solution containing lysozyme (2.0 mg/ml in 10 mM Tris-HCl, pH 8.0) and incubated at RT for 15 min. Alkaline-SDS (10 ml) was added to the mixture and again incubated at R T for 10 min after mixing the contents gently by inverting the tube. Post-incubation, chilled sodium acetate solution (7.5 ml) was added and the contents were incubated on ice for 15 min. After incubation, the mixture was centrifuged at 10,000 X g at 4°C and processed in the similar fashion as described above upto addition of isopropanol. The DNA pellet was resuspended in 500 Jll TE containing 20 Jlg/ml RNase and incubated for 1 h at 37°C. Plasmid DNA was then extracted as described above. The DNA pellet was air-dried and finally dissolved in 200 Jll ofTE

Large scale plasmid DNA isolation

EROD

TheO2consumptionofthetissueswasmeasuredbymanometrictechniquesinaWarburgconstantvolumerespirometer(Gallenkemp,England)aspertheproceduresgivenbyUmbreitetal.(1959).Thecontrolandeffluentexposedfisheswerekilledandthebrain,gill,muscle,liver,heart,kidneyandair-breathingorganswereisolated.ThetissueswereslicedandplacedinWarburgflasks(60-80mgtissuesflask)containing2.5mloffishringersolutionwithphosphatebufferatpH7.5asthesuspensionmediumforthetissuesand0.2ml15%KOH.Temperaturewas28°CduringO2uptakedetermination.Inordertocomparedatafromthedifferentseriesoftreatment,arespiratoryindexwascalculatedusingthefollowingformula:KO2treated-KO2controlr=100-------------------------------------x100KO2controlWhere,K=O2consumptioninpi/100mgwettissue/hrThisindexindicatespercentrespirationoftreatedtissuesrelatedtothecontrolvalues ofthesameseries.

TissueRespiration

incubator until the cells were 60% confluent. For each transfection, 1-2pg of DNA was diluted in 100 pi serum free media. Also, lOpl of lipofectin reagent · was diluted in 100 pi of serum free media and allowed to stand at room temperature for 30-45 minutes. The two solutions were combined, mixed gently and incubated at room temperature for 15 minutes. The cells were washed once with 2ml of serum free medium. For each transfection, 0.8 ml of serum free medium was added to each tube containing lipofectin-DNA complexes. The complex was mixed gently and overlaid onto cells. The plate was incubated for 4-6 hrs in a CDl incubator. The medium in each well was replaced with serum containing medium and the cells were further incubated for varying periods of time at 370C. The concentration of lipofectamine 2000 was used in the ratio 1:2 or 1:3 with DNA. The Rzs and Dzs were either co-transfected with the plasmid DNA of interest or when required to be transfected alone then pBSK+/-was used as carrier plasmid for better transfection efficiency. In order to ensure uniform transfection efficiency a reporter plasmid DNA (pSV -~ gal, Promega) was used

Transfection of cell lines used was carried out u5ing lipofectin reagent (Invitrogen, U.S.A.). In a six well plate 10 s cells/ well were seeded in 2m1 medium supplemented with serum. The cells were incubated in a CD2

Materials & Methods dried. These were counted directly to determine the total counts. In duplicate tubes, 1pl of the diluted probe was added to 100pg of carrier nucleic acid (tRNA or Herring Sperm DNA) in a total volume of 100pl. To this 500pl of ice-cold 5% TCA was added, mixed thoroughly and incubated on ice for 15-20 min. Glass fiber filters were wet (in duplicate) properly with 5% TCA and then these samples were applied on to them under vacuum. The filters were washed twice with 5ml of chilled 5% TCA and then air dried after rinsing with 2m1 of acetone. All the dry filters were inserted into scintillation vials containing scintillation fluid and the counts were taken in a liquid scintillation a-counter (LKB Wallac, 1219 Rackbeta, Sweden). The percentage incorporation, specific activity and the total amount of RNA made was then calculated according to the standard procedures. % incorporation =Incorporated cpm X100 Totalcpm Total RNA made (ng) = % incorporation X 338 Specific activity of probe = Total cpm incorporated p.g of RNA synthesized Cell culture media and cell lines: All the cell lines were grown and maintained in Dulbecco' s modified Eagle's medium (DMEM) with 10% Fetal bovine serum (FBS) and 1% antibiotic-antimycotic (penicillin, streptromycin and amphotericin B). The cells were maintained at 37<>C with 5% C02 in a humidified CD2 incubator (Nuaire-IR Autoflow CD2 Water-Jacketed incubator). Transient transfection

After PCR, 1 μl of Dpn1 enzyme (10U/μl) was added to the amplification mix and incubated at 37°C for 6hours. After that, 10ml of the amplification mix was taken to transform Dh5a cells. Positive clones were selected after confirming the sequence of plasmid DNA

The PCR parameters were as follows

The reaction mix included 2ml of PSKll(39+) (50ng) containing wild type K-Ras cDNA , 5ml 10x buffer, 20pmoles of primers , 1ml of 10mM dNTP mix and 1ml of deep vent polymerase (NEB).

The following K-Ras-ras mutants were generated by site directed mutagenesis according to the protocol described in QuickChange site directed mutagenesis kit (Stratagene). The primers used are shown in the following table

Site Directed Mutagenesis

Denaturation 95°C 1 min After 30 cycles of PCR, the final elongation step was carried out again for 10 min at 72°C

The PCRs were normally performed using a PCR amplification kit from Fermentas/Sigma (USA), following the company's protocols. Approximately, 10 ng of chromosomal or 1-2 ng of plasmid DNA was used as template in a 50 μl reaction volume containing 0.2 mMM of each dNTP, 20 picomoles each of forward and reverse primer and 0.5 units of Taq DNA polymerase. In some cases, freshly streaked E.coli cells from a plate were resuspended in 50 μl of sterile Milli Q water to get a cell suspension (~ 109 cells/ml) and 10 μl from this was used as the source of DNA template. The samples were subjected to 30 cycles of amplification and the typical conditions of PCR were as follows (although there were slight modifications from one set of template/primers to another): The initial denaturation was done at 95°C for 3 min and the cycle conditions were as given below. Annealing 55°C 1 min Elongation 72°C (1 min/kb of DNA template to be amplified)

Polymerase chain reaction (PCR)

Most chemicals were obtained from commercial sources. The sources for some of the fine chemicals used in this study are given below. Most of the chemicals such as amino acids, antibiotics, sugars, IPTG, ONPG and X-gal were obtained from Sigma Chemical Co. The media components for the growth of bacteria were routinely from Himedia. The materials used in the recombinant DNA experiments such as restriction endonucleases, T4 DNA ligase, DNA polymerases for PCR amplification and DNA size markers were obtained from companies including New England Biolabs and Fermentas. Quiagen or HiPura Kits used for plasmid isolation, purification of DNA fragments. The oligonucleotide primers used in this study were mainly synthesized on order by Ocimum Biosolutions or MWG Biotech Pvt. Ltd

Chemicals

1mL medium were stained with 1p.L of the dye (1ng/mL) and incubated at 37°C for 10 min in the dark. The unbound dye was washed off with either PBS or medium and cells analysed by flow cytometry or microscopy. Nonylacridine Orange (NAO): NAO (Molecular probes) is a probe which interacts specifically with non-oxidized cardiolipin, a lipid that is exclusively localized in . the inner mitochondrial membrane (Petit et al., 1992). A stock solution of 100p.M was prepared in DMSO and 1p.l (1nM) was used to stain 107 cells in 1mL medium for 10 min at 37°C. The excess dye washed off with PBS and cells were fixed with 4% formaldehyde for 3 min. Subsequently they were analysed with flow cytometry or microscopy

Mito Tracker Green®: Mito Tracker® Green (Invitrogen, Carlsbad, CA) is an agent that interacts with mitochondrial lipids and is essentially non-fluorescent in aqueous solution, only becoming fluorescent once it accumulates in the lipid environment of the mitochondria. Since the entry of the dye is not dependent on the mitochondrial membrane potential, it can be used to measure/ compare the mitochondrial mass of cells both in live as well as fixed states. MitoTracker Green® stock solution was prepared in DMSO (1pg/pL) and stored at -20°C. To stain cells

Assay for measuring mitochondrial mass

The PCR products generated using protocol mentioned above were purified using QIAquick PCR purification kit from Qiagen (Hilden, Germany) as per manufacturer's protocol. Briefly, S volumes of buffer PB was added to 1 volume of PCR sample. This was applied to QIAquick spin column and centrifuged at 10,000 x g for 30-60s. The flow-through was discarded and the column washed with 0.7SmL of buffer PE. After discarding the flow-through again, the column was dried by a quick spin. The DNA was eluted using 30-SO J..lL of elution buffer (Buffer EB (10mM Tris-Cl pH 8.S)) or alternatively in nuclease-free water. The concentration of the obtained DNA was estimated by measuring the absorbance at 260nm (A26o) and using the known formula: DNA concentration = A260 X SOX dilution facto

Purification of PCR produc

were maintained in log-phase by continuous passaging in fresh YNB medium every 4 h

For phosphate starvation,yeastcells grown to log-phase in YNB medium were harvested,washed with water,transferred to either regular YNBor YNB medium lackingphosphate and were grown for 16 h at 30 ̊C. Cells cultured in YNB medium

Phosphate starvationof yeast cells

.coliBW23473 electro-competent cell aliquots werethawed on ice and mixed with 1-2 lof plasmid DNA. Mixture was pulsed with the Gene Pulser®electroporation apparatus(Bio-Rad) at 1800 Volts with 25 μF and 200 Ωcurrentin a chilled0.1 cm electroporation cuvette(Bio-Rad). Immediately after successful pulsing, 1 ml LB medium was added to the cuvetteand suspension was transferred toa 1.5 ml sterile centrifuge tube. Cells wereincubated at 37°C for 1 hwith shaking and further plated onLB plates containing kanamycin(30μg/ml). Positive colonies were inoculated in LBliquid medium containing kanamycin(30μg/ml)for plasmid isolation