0022THE

1554/79/2701-0087$02.00/O JOURNAL OF HISTOCHEMISTRY

Copyright

© 1979 by The

AND

H#{252}tochemical

Fluorescence

Decay DONNA

Abteilung

J.

Molekulare

(D.J.A-J.,

Inc.

Analysis Chromosomes

ARNDT-JOVIN,

Biologic,

G.S.,

Vol. 27, No. 1, pp. 87-95, 1979 Printed in U.S.A.

CYTOcHEMISTRY

Society,

T.M.J.),

in Solution Stained

SAMUEL

A. LATT,

Max-Planck-Institut

Clinical

and with GEORGE

STRIKER

f#{252}r Biophysikalische

Genetics

Division,

Harvard

Medical

Children’s

Hospital

School,

Received

Boston,

for

in a Microscope Quinacrine AND

Chemie,

Postfach

Medical

Center

Massachusetts

publication

July

24,

of DNA

THOMAS

M. JOVIN

968,

G#{244}ttingen,

and

D-34tX

the

Department

and

W.

Germany

of Pediatrics,

(S.A.L.)

1978

The fluorescence decay properties of a variety of DNA and chromosome preparations stained with the intercalating dye quinacrine have been determined with a single photon counting instrument adapted for solutions or slide-mounted specimens in a microscope. Multicomponent analyses of the decay curves with the method of modulating functions revealed at least two (and probably three) components for the free dye at neutral pH with r’s of approximately 1, 3 and 8 nsec. The total emission intensity and fractional contribution from the longest decay time increase at higher pH’s. Binding of quinacnne to DNA containing G leads to fluorescence quenching, while binding to DNA containing only I or A as purine residues enhances fluorescence. The decay curves in all cases are characterized by components with longer lifetimes than those of the free dye. Representative lifetimes for poly[d(A-T)] are 1, 8 and 26 nsec. Similar values were obtained for bacterial DNA of differing A-T content. However, the contribution to the total emission made by the longest decay process is related directly to A-T content, while the total emission varies approximately with (A-T)4. Preparations of human and drosophilid nuclei and chromosomes on slides stained with quinacrine showed decay curves similar to those for DNA in solution. Consideration of the content of satellite DNA of known composition (and in some cases sequence) indicates that the emission properties are correlated with A-T content and the distribution of interspersed CIGC base pairs. Other factors such as nucleoprotein and higher-order structure may be of less importance. We conclude that the brilliantly fluorescent regions of quinacrine-stained chromosomes may consist of regions with a high A-T content, including clusters relatively free of G-C base pairs.

The

characteristic

mosomes

stained

niques

introduced

karyotyping abnormalities tion and

of

chro-

properties

according

to

tech-

steady-state

Caspersson

content

patterns

(3)

revolutionized and and

decay

genetic absorp-

that be

and for

Physical

biochemical

7,

quinacrine 14,

20,

affected

DNA

samples and

specificity

(14, pare

18, 19). However, few cytologic and solution of fixation

and

to several

banding pH

and

analysis

and

smears

that

features

of the

provided

attempts studies

condensation

optical

However, of salt, proteins DNA

The yields difficulties.

binding

information stability have and

may

of the

of the

DNA

If the

net

of dye-DNA

is

the

of decay

data

to

decay

with

A-T

bacterial

slides

analysis

and

The

measurement

does

not

require

time

of

content

different

and

was

less

than the quantum yields. Similar obtained using single component

for

microscope

power.

a dynamic

average

gradually

to base composition have recently been on

interaction,

discriminatory

et al. (14) of DNA-bound quinacrine analysis of the fluorescence decay

that

increased

taken by Duportail amounts of the

by

of several

DNA Andreoni

DNAs

in solution

et al. in solution

and

as

( 1). A twowas

et al. (5) who saw no change in the two decay times measured for the

underrelative various

DNAs.

complexes

of emission preparations

modes lack

however,

indicated

component

regarding

as factors

several

fluorescence,

be

The

been made to comto assess the impor-

determination of cytologic

of

complexes sensitive results

indicating

thermodynamic

phenomenon. and quantum

25).

evito DNA

variety

from

measurements

Initial studies by Latt using single exponential

is related

data 12, 13). of reversible quinacrine

have

the

the banding intensities

24,

a wide

structural of these

measurements

different

21,

by

tertiary

biophysical

banding

treatments

reviews

and

derive

which provides additional resolution knowledge of absolute concentrations.

curves

enzyme

secondary

subject

al.

of polymorphisms Other fluorescence

abundant (4,

can

proteolytic

tance

et

were developed subsequently (for review, see 6, 12) additional questions as to the origin of the bands

to suggest

involved

patterns

mustard

by

(4, 5, 7, 13). There exists

A-T

banding

quinacrine

and the study of eukaryotes.

stains raised

dence

fluorescent with

lifetimes variety

in is

fluorescence

goal

of this

solution fluorescent

measurements species

present

the

measured:

paper

is a comparison

of the

fluorescence

of cytologic preparations of chromosomes of sources measured in a microscope with are

fluorescence (a)

as

a function

87

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on April 29, 2015

data

from a from

so as to determine whether the present in both types of samples. decay of

parameters pH;

(b)

in

of solution

same We

quinacrine complexed

88

ET

ARNDT-JOVIN

to

a variety

DNA;

of synthetic

and

(c)

Drosophila

and

have

analyzed

been

polymers

in a microscope human

DNA for

and on

and

two

and

naturally

cytologic chromosomes. three

2-hydroxyethyl piperazine-N’-2-ethane-sulfonic acid (HEPES), pH 7.5 at 20#{176} or, in the case of unbound quinacrine, at the pH’s indicated. Human peripheral lymphocytes were cultured in Dulbecco’s minimal essential medium (MEM) supplemented with 10-20% fetal bovine

occurring

preparations

of

The

exponential

AL.

data compo-

serum.

nents.

Cells

MATERIAL

AND

METHODS

Quinacrine was obtained as the dihydrochloride from Sigma Chemical Co. (St. Louis, Mo.) and shown to be virtually free of contaminants by thin layer chromatography on silica gel. Polynucleotides were synthesized using Escherichia coli DNA polymerase and calf thymus terminal transferase. DNA samples were the kind gift of Dr. W. Muller except for calf thymus DNA which was purchased from Sigma Chemical Co. (type V, highly polymerized). All samples were extensively

and

dialyzed

tinction coefficients were Solution measurements

deproteinized

taken were

free

of nucleotide

Fluorescence

Lifetimes

Calf thymus Poly[dA-dT] Poly[d(A-T)] Poly[dI-dC] Poly[d(I-C)1 Poly[dGdC]h Poly[d(GC)]h Quinacrine

,.y1

DNA

Ex-

0.01 M N-

ofQuinacrine

or polymerh

DNA

precursors.

from the literature. performed in 0.1 M NaCl,

Bound

(nsec)

extract

TABLE Ia to DNA and

(ruec)

i

were

harvested

3 days

between should N(

thus

= mean

(r2)

c)

weighted

high

average

number

counting

noise.

kidney

Polymers

(Tu’o-Component

%

fce

(ruse)

i2

Analysesj fce

(r2)’

(c)’

15 18

5-6 5-6

40

21-24

60

1.34

62

20

21-24

80

1.94

500

3.1

19

5-6

26

24

74

2.02

412

1.4

10

72

1.34

20

28 21

13-17

3.2

2-4 5-6

22-24

78

1.14

190 280

0.1

13

1-3

18

16-21

82

4.41

14

0.1 1.0

13 6

1-3 3

18 48

15-20

82

2.34

44

8.5

52

2.5

86

squared

residual

of counts Under

of a saline

0.57 2.9

experimental and calculated approach the value of 1 in the =

addition

phytohemagglutinin.

containing

a Excitation of an Ortec free-running N2 lamp at 1 atm. was filtered by a Corning bandpass 7-51 filter Emission was collected above 470 nm. Analyses were performed on 500 channels of data. 1 Quinacrine concentration 1 tM, polymers 50-150 M. rte = relative total emission intensity normalized to unbound dye. These values are not proportional effects upon binding of the dye are not considered (see 13, 14). d (T) = average lifetime = fce . e fce = fractional component emission = a-r/aei’e where a is the amplitude.

I

after

Slides were prepared as described previously (13). Slides containing Samoae leonensis brain cells or Drosophila virilis nuclei were the generous gifts of Dr. John Ellison and Dr. Gerald Holmquist, respectively. All microscopic samples examined were stained with quinacrine at pH 5.5, washed with water, and mounted in 0.01 M NaC1, 0.005 mM N-2-hydroxyethyl piperazine-N’-2-ethane sulfonic acid (HEPES) buffer, pH 7.0. Instrumentation: Time dependent fluorescence measurements were done by the method of single photon counting (26) using an Ortec (Oak Ridge, Tennessee) 9200 spectrophotometer modified as follows. The light source was the Ortec 9352 lamp either in the free running mode at 1 atm N2 for the microscope experiments and early solution studies or at 3.5 atm N2 for increased light above 400 nm for bean

!

=

counts), limit

and

of an

per channel

these

(.x)12/x1,

the

weighting

accurate

analysis

!

=

conditions,

where

the

x1.

iow

factor of data

values

goodness

number

n is the

for

of fit

1/x

of channels,

is the

devoid both

reciprocal

of systematic ( c ) and

is difficult

variance

derive

to estimate.

oriented

with

to quantum

yield

(i.x), is the residual

error.

( r2)

and

due Our

from

to

Conversely,

values

since

counting

with

The

to minimize

relatively

of ( r2)

i (difference

statistics.

seek

at 35.3#{176}.

hypochromic

of channel

analyses data

a polarizer

>

few I for

(r2) r2).

counts

and

measurements

with good counting statistics (large (c)) are indicative of a poor fit (e.g. due to an insufficient number of components) and/or systematic error in the data. In this and other tables, one channel equals 0.127 nsec. h The quenching of these polymers is so extensive that the analyses are subject to large error and variation. In addition, a possible contamination with a DNA producing a highly fluorescent quinacrine complex (e.g. the template used in the synthesis) cannot be excluded.

Fluor or polymerb

DNA

Poly[d(AT)]e Clostridium

Proteus Bacillus

acidurici

mirabilis subtilis

a Excitation as in Table ,, C

d

..

escence

Lif etimes

of

Q

uinacrin

(A-TY

(A-T)4

rte”

1 0.7 0.6 0.55

1 0.24 0.14 0.098

1

at 435 ± 5 am with

Ortec

lamp,

e Bound (r) (nsec)

18 14 14 12

.2 .15 .12 3 atm

N2. Emission

TABLE

lb

to

a nd Poly[ d(A-T)J

T

DNA (nsec)

fce %

1.1 3.0 3.3 2.6

6 29 29 35

(T hree-Co

(nsec)

i2

fce

7.9 11 13 11

at 516 ± 5 nm. Analyses

33 36 45 39 performed

%

mponent i:

Analysis)”

(nSeC)

fce %

(r.2)

(c)

26 27 30 27

60 34 25 26

2.71 1.20 2.34 1.26

1040 566 2326 437

on 1000 channels

of data.

Abbreviations

la.

Quinacrine concentration 2 tM; polymers Fractional A-T composition. Total emission intensity relative to that Measured with excitation light polarized

at 200 M;

0.01 M HEPES,

for poly[d(A-T)]-dye complex. at 35.3#{176}. (See Fig. 2.)

pH 7.5, 0.1 M NaCI. For values

relative

to free dye at pH 7.5, multiply

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on April 29, 2015

by 3.2.

FLUORESCENCE

DECAY

ANALYSIS

OF

89

QUINACRINE-DNA

(I)

z 0

C-)

a uJ M -J

0

z

350

700

CHANNEL FIG.

nsec/channel.

1. Nanosecond Data

lamp;

open

curves

are

triangles, given

decay curves for quinacrine bound recorded as described in Materials

were

Bacillus

in Table

subtilis

DNA;

x, Clostridium

An RCA 8850 photomultiplier and an Elscint (Haifa, Israel)

snap-off

replaced

discriminator

amplifier/discriminator yses

were

filters

performed

given

in

below

the

Ortec

(Results).

In some

of emission anisotropy on the decay izing the exciting light at an angle correct for drifts in the lamp profile between

the

cycled

filter changes) every lOb flashes (about LUDOX (E. I. Du Pont de Nemours Del.) was used as a scattering solution

response

function

ofsuccessive

using

lamp

A Zeiss universal Inc., Oberkocken, running

N2 lamp

51 bandpass

sample

cases,

propnate containing mington,

data

filter

and

identical

were and

sample

scattering

excitation

collected

in the

records

epi-illumination W. Germany) imaged through onto

acidurici

a dichroic

(RCA, STD N-2 and 463 height analand records interference

possible

influence

curves was eliminated of 35.3#{176} to the vertical during data collection

matically

Microscope

454

in later experiments. The pulse a Fabritek 1074 (Madison, Wisc.) 1 108. The excitation and emission

on the Univac are

(usually

fluorescence was equipped a collimating mirror

solutions

with

(with

ap-

1 mm). A cuvette and Co., Inc. Wilto collect the lamp

and

more

by polar(1 1). To we auto-

emission

filters.

conventional of3O-60

mm

manner

to

the

RCA

8850

microscope (Carl Zeiss with the Ortec free lens and a Corning 7peak

reflection

between

photomultiplier.

Background

data

were

ob-

tamed on regions of experimental slides not containing cells. A microscope slide covered with reflective foil was used to reflect the lamp pulse to the photomultiplier using a broad bandpass Balzers K-i filter (Liechtenstein) centered at 400 nm as emission filter. Multicomponent

the Univac

data

curves

using

are

overlaid

alternating

DNA;

+,

analyses

1 108 of the Gesellschaft

were

carried

out

fuer Wissenschaftliche

interactively

Datenver-

arbeitung

poly

with

1-mm

flash

[d(A-T)].

normalization and

The

on

(Goettingen,

tions

to peak

sample

results

records.

Open

of exponential

height, circles,

analyses

0.127 flash

of these

(Striker,

introduced

Germany)

personal to

using

a method

communication).

nanosecond

decay

analysis

of modulating

Modulating by Valeur

func-

functions were and Moirez (22,

23).

Interpretation of the results was made in terms of the fluorescence lifetimes (T,) and amplitudes (a,), the normalized product a,T/Ea-,r, (fractional component emission) and (T) (the average lifetime) for each component. The fractional component emission (Ice) in the tables

represents

the

relative

contribution

made

by a particular

decay

component to the steady-state fluorescence (26). The average fluorescence lifetime, (r) = ).fce, . r1 , is the first moment of the (multiexponential) fluorescence decay curve and constitutes a useful single parameter representation of the latter. In addition, the relative total emission (rte) of each sample was determined by measuring the photon count rate (26) under conditions of

duration).

350-400 nm. The light was focused on the specimen with a bOX Neofluor phase objective through a quartz cover slip. The emission was collected through the same objective, and passed through a dichroic mirror with transmission above 410 am and a 470 nm barrier filter

Decay

Methods

lb.

later solution experiments. Camden, N. J.) was used

analyzed

to DNA. and

dark

noise

suppression

overlap.

These

values

-quinacrine

(or

were

subtraction)

normalized

and

to those

absence

of

pulse-

for the poly[d(A-T)]

complex.

RESULTS Solution studies: The fluorescence quantum yields for a variety of synthetic DNA species are given in Tables I a and here ratios tually The binding

are

for

DNA

concentrations

(P/D) greater all of the added fluorescence to synthetic

than dye intensity polymers

50 50/1 should

tM

and

conditions be bound.

phosphate/dye under

of quinacrine lacking dG-dC

cluding dI:dC). The relative increases are 5-fold; the dye absorptivity in the excitation is decreased by a factor of approximately

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on April 29, 2015

lifetimes and relative polynucleotides and b. The data presented which

vir-

increases upon base pairs (in-

on the order wavelength two upon

of 4. to region binding

90 (14).

ET

ARNDT-JOVIN

Quinacrine

bound

dC is almost to DNA to a lesser

completely samples differing

to polymers quenched widely

containing

only

dG

and

The

DNA

whereas quinacrine bound in A-T content is quenched

extent.

AL.

fluorescence are

decay

correlation

shown between

processes.

The

in

curves

Figure a higher

for

complexes

with

1. Visual inspection A-T content and

quantitative

analysis

of such

data,

various

reveals a slower decay however,

is

(/l

NS

20

i72

CHANNEL NRNSEC

1. 1132+000

7.9591+000

TRU (C)

8. 9021

6. 2651

AMPL INTENS

7. 9519-003 6.3793-002

+000

2.6283+001

+001

2. 0609’002

5. 701-003 3.3519-001

3. 1873-003 6.0102-001

0.

0.>< Li

cz

0

I_

0

I-------1-

113

23

I

35’t

WTD.

1

I

72

I

590

1

I

708

I

I

82

I

I

9i4

DEVIATION

FIG. 2. Analysis of fluorescence lifetimes of quinacrine bound to poly d(A-T). Conditions are as in text. Top panel displays the flash, convoluted data curve and the simulated curve overlaid on the data for the calculated r’s and amplitudes given below the panel, 0.127 nsec/channel. The middle panel is a plot of the weighted residuals from the difference in the simulated and experimental curves. The bottom panel is an autocorrelation function of the residuals. The high frequency periodic noise is due to radio frequency interference from electronics associated with the instrumentation. INTENS is equal to (fce) fractional component emission in Table lb.

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on April 29, 2015

FLUORESCENCE

DECAY

ANALYSIS

OF

TABLE Quinacrine pH

(nsec)

(n.sec)

fce %

mes

(nsec)

II as a Fu nction fcc %

ofpH” fee %

rteb

r./)

(c)

4.4

2

0.9

42

3.3

50

7.4

8

0.97

2.15’

2470

25 4

3.5 3.0

54 25

9.0 8.3

21 71

1

1.33”

1745

1.9

1.26’

2.8

20

8.6

80

2.2

1.26

528 954

(T)

i

7.5

4

0.7

8.8

7

1.3

10.0

7

a Dye concentration 2 M, buffer Abbreviations as in Table 1. Analyses F,

Lifeti

91

QUINACRINE-DNA

Comparable

value

values

were

0.01

M HEPES,

performed from the

obtained

r2

0.1 M NaCl. 1000 channels

on peak

Excitation

435

of data. of emission

intensities

Ti

(nsec)

± 5 rni,

spectra

Ortec

obtained

lamp

at

3 atm.

N2.

in a spectrofluorimeter.

Emission

516

± 5 nm.

Normalization

is to the

pH 7.5.

for

A two component The two component .. The two component

analysis

(.

(I

gave

r (fce)

analysis

gave:

analysis

gave:

values

1.9 nsec 3.1 nsec

1.6 nsec

of:

(58%), (35%),

(66%),

7.1 nsec 8.7 nsec

4.9 nsec

(42’/r). (65%).

r,2) r2)

(34%). was was

(r,2)

was

2.93.

2.23. 1.22.

(I)

F-

z

: 0

C-)

a Ui M II

-J

:: 0

z

500

CHANNEL FIG. 3. Fluorescence lamp; X, pH 4.43; open

conditions

decay triangle,

are as in Table

the

due

greater

than

three)

exponential

presence

lb

reveals

a fairly

relative total emission T content, an observation Rigler position. the

a greater ponents, rable. equals An

and

of

at least components.

curves

of DNA

relative although The

2530

linear

0 for (AT)4

component

fact the unbound dependence

ns)

with

contribution the individual

fractional is in

at

two

various pH 8.8;

linearly dye. of the

(and

correlation

the fourth power made previously

(20). The r is a relatively weak Closer examination, however,

decay

(T:I

for quinacrine 7.5; open diamond,

pH’s.

Data

pH 10.05.

+,

are overlaid Data were

as in Figure 1, 0.127 taken using alternating

nsec/channel. 1-mm flash

Open circles, flash and sample records;

III.

difficult

Table

to

curves pH

higher from

between

of base a tendency

content

the longer lifetimes are

requirement

Aand cornfor

to contain lifetime cornquite compa-

emission

for the

longest

time

related

to A-T

content

and

is consistent

with

four

fluorescence is not well

A-T

enhancement. established,

base

pairs

The however

in

order

to

stoichiometry (14, 20), probably

at

to the complication of excluded binding effects. All of the data in Table I were fit using an analytical procedure allowing for two or three exponential decay com-

ponents. relation

The quality function and

representative

analysis

In view of the complexity be regarded as unique number a

adjacent

due

cision.

emission

for

potentiate saturation

the

of the relative by Pachmann

function reveals A-T

probably

polymer

of exponentials Over

for

were judged by the autocoranalysis of the residuals.

poly[d(A-T)]

required we

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on April 29, 2015

is shown

in Figure

A 2.

of the system, however, a fit cannot but rather indicates the minimum of 4 years,

a period

solutions

of the fits a statistical

have

to fit data using

obtained

of a certain

various reproducible

flash

lamps

preand

two-com-

92

ET

ARNDT-JOVIN

ponent

analyses.

averages higher

of additional components light levels (see Table Ib).

analyses

the

component The

DNAs

by the

we

reproducibility is not

samples. ring

However,

of the

good

residuals

not

with

thus

indicate

and

possibly nonexponential, At least part of the

of Quinacrine

the

(T)

(ruec)

Human 46,XX Human 46,XYqb Human 46,XY Drosophila virilis’

10

Samoae

15

leonensis

times

for

the

highly

are at

tion and

occur-

fce

Ti

(nsec)

9 9

2-3

9

2-3

(%)

3

50

3

55 49

14-18

48

11-15

3

24

fce

18

16

19

of the

of multiple ionic both the quantum

unbound

dye

species. yield

undergo

large

of pH. Table II and Figure 3 depict the decay curves, respectively, for

Massari et al. and absorption

changes

in the ponents

pH. The longer pH as is especially

be

(%)

r,

50

1.6

54

C

45

0.86

40

51

1.03

45

52

1.4

920

76

1.02

140

a Excitation was made with Ortec free running lamp at 1 atm. by epi-illumination on microscope slides using a Corning 7-51 bandpass filter. Emission was collected above 470 am. Analyses were made on 500 channels of data. b Human male lymphocytes lacking polymorphic quinacrine bright staining of the Y chromosome. C The fluorescence decay data contained considerable scatter. Best analyses were obtained by limiting the fitting program to the response curve after 4.5 nsec beyond the excitation peak. d Scatter contributions again limited the analysis to the response curve starting 2 nsec beyond the excitation peak.

from

the

values

of

at neutral at higher (

studies: of cytologic

has already made decay

been observed) measurements

fluorescence intensity of the analyses were

The problems

data a)

To test samples

the

N2 lamp b) scattering

decay cornapparent

effects of fixation and (those in which banding lifetimes, Since

we the

was low from these preparations most done on metaphase cells and interphase spread in Table

metaphase III.

taken on the microscope the limited light levels

running

the dye. It is species coexist

on the quinacrine directly in a microscope.

than on the Data are given

optics,

lifetimes cover

r).

Microscope condensation

nuclei rather themselves.

of

which

state. might

free dye alone predominate

as a func-

fluorescence four pH’s

of additional,

Preparations”

r2 (nsec)

presence that

protons fluorescence)

III

to Cytological

to the showed

the range of the three titratable obvious that several ionic (and

as judged

presence

due (16) spectra

middle

quenched

to naturally components

in the excited decay phenomena

Bound

DNA

the

bound three

processes observed

these

time

for

TABLE

Lifetimes

decay

lifetimes

fit well

that

seen with data taken In the three-component

especially

quinacrine are

believe

AL.

focussed

through

of the

slide, and c) differences the flash and emission

the

excitation

chromosomes

suffers available

from using

non-quartz pulse

several a free

microscope

by the

cytological

in the response signals

due

time to photons from to widely spaced excitation

and emission wavelengths. The first problem limits the her of components which can be fit with accuracy to no than 2. The scatter contribution to the emission decay was corrected for by blank subtraction in some cases analysis was performed nent fit for the scatter

using one signal. These

component corrections

nummore curve

or the

of a 3-compowere checked

(1)

F-

z 0 0

a Ui

M I-4

-J

0

z

250

500

CHANNEL Fluorescence decay curves microscope as described in Materials triangle, nuclei from a normal human FIG.

4.

for quinacrine bound to cytologic samples taken by microfluorometry. Decay curves were taken on the and Methods and data were analyzed is in Table III, 0. 127 nsec/channel. Solid line, flash lamp; open XY male; x, nuclei from Drosophila virilis; +, nuclei from Samoae leonensis.

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on April 29, 2015

FLUORESCENCE

DECAY

ANALYSIS

OF

93

QUINACRINE-DNA

(0

n-,. 10

-

(CL

,

cn r I-

.

1

H

tJ

I H

F4:,

I

U-’-

‘PS

‘

\

t.

I

\\

4

-

Ck

-.

Li

!.

‘.

.

.

.

:ci

1)

I

JPNSEC I

iRl

1. O5

:

C

RMF’L

i.

[r1-E1,’j,.,

.::.

1

.‘[

“.

.

::

..

I#{149}i i

iE

U

,. ‘.o-

C;;’,,

t:(IJ73-.l:H:ti

r

..

j

O[J .3 #{182}5’:’’7-C1lLJ

(1

JItLi[

i’i

liii

J

.I

h

.

,,

id i1Ii

i

i

i

tii1iii

J .J

,11j

(I

1

(fi.

Ii

-

V,1Ir

FIG.

5.

Analysis

in Figure 4 on two-component

. ‘-

I

. 0-.

of fluorescence

.

vr’”

lifetimes

the microscope. The panels fit for any of the microscope

.

.

.

I

.

r’

.,-“‘ k

I

of quinacrine are

the data.

same

!..

.i&

‘‘‘‘

bound

-

to single

as in Figure

cells

2, 0. 127

of Samoae nsec/channel.

leonensis The

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on April 29, 2015

on a microscope three-component

slide. fit was

Data not

were better

taken than

as the

94

ET

ARNDT-JOVIN

by

starting

the

analysis

as to minimize pendent plier

the

temporal was

The normal

with

perhaps

calf

decay female

1%

of the

time) the

offset

channel

total

between

the

flash

which

opti-

for nuclei from both showed comparable measured. fluorescence

cellular

Since the inis associated

DNA,

these

results

expected. The relative contribution of short and times were similar to those determined in solution thymus

human To

DNA,

DNA. examine

which

has

base

base

composition

in cytologic

ture

of the

in

accordance

long of

yinosinate

nuclei

and S. leonesis. inant satellite

The DNA

per

we

from

former species,

two

organism one with

heptameric

repeat

(8),

of quinacrine

measured

drosophiids,

D. virilis

nuclei with brilliant a satellite with only

quinacrine A-T base

approxi-

complexes

originating satellite corn-

average

over

lifetime component

synthetic

Figure

consistent regions

4 shows

with the attribution according to the solution

the

three cytologic preparations. ysis of the decay curve for

superimposed

Figure S. leonesis

decay

of

this mea-

curves

for

5 is a representative nuclei.

anal-

present

report

fluorescence decay solved spectroscopy the rial

mechanisms such as cells.

sites

of

we

of fluorescence Such systems

ground

and

measurements culty

illustrates

cannot

was

other

DNA,

than

problem section. An dye by

as

et

Andreoni

quinacrine

apparatus

light

binding

source

al.

(1)

were analyzed for only an inherent restriction. of

which

we

are

aware

in

1976.

one The in

discussed

the

to

previous

of N2 laser was

Recent

pumped

this

of

instrument

decay but this is not report in the literature decay

spectroscopy

This

of quinacrine

appearance

depends

on

was

14,

quinathe

na-

tables

20,

and

25).

The

quenching,

deox-

deoxyadenylate

to

due either site and/or

no

consistent

a

to primary secondary

variation

G-C content quenching

of sequence of fluorescent we have

probably

dye

sites.

binding

in ap-

(14, 18). in the case

polynucleotide the intensities (A-T)4, a finding

dependence

to all DNA

of a longer composition

re-

reflects

an

Holmquist

However, the dye,

(Table

the existence the possibility

samples

lifetime and

polymers

(10)

and

both

in

natural

of at least two of both dynamic

state emission

component

sequence

Ia)

is characterized

decay

which the

DNA

of

(Table

protonation and static

processes, and lead to decay

case

Ib).

states quenching

the superposition curves for the

of

which are difficult to decompose. fluorescence decay of the alternating can be fit quite well by three lifetimes

(1, 8, 26 nsec; Table gave fits of comparable of more components.

Ib,

approximately the

Fig. 2), none of the bacterial DNA quality, an indication for the existence A characteristic finding, however, is the

linearly case

with a lifetime the proportion with

of the

A-T

as well,

(8),

one

can

calculate

where i. Doing

DNA

of S.

leonensis

mean

value

is

of

this

close

to

Due

to the

the

low

to have value

ofO.13

photon

levels

an

decay

curves from

and

their

those

(A-T)4

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on April 29, 2015

for

(

T

the

which

the

is

DNA.

data

do

of quinacrine However, the

) values

leonensis

from

human

microscope,

S.

and

ofO.17 the

the decays chromatin. the

of

appears

value

to

fluorescence

satellite

virilis

for

corresponding

derived

equal content

ofthe

A-T

calculated

in the

quantity

85% D.

is com(17) and are known

nucleotide

the

The

not permit discrimination between bound to D. virilis or human 46,XY different

(A-T)4

that by

is 0.47.

information

very

we found

of which

A-T residues sequences

fractional

contributed

(A-T)4

40%

only satellite

a mean

a, is the

of 25-30 nsec (in the of which increases

content.

S. leonesis

of satellite DNA containing case of D. virilis for which

a,(A-T)41/a,, component

of

quin-

acrine-DNA complexes Thus, although, the copolymer poly[d(A-T)]

sequence

reported

measurements with

the

prised in the

primarily

in

Binding by

In

from

on cytologic preparacomplexes. The

DNA

which

diffi-

in the

5,

and

that to

presence of a component three-component analysis),

static

intensity

limitation,

exponential only other

matebinding

experimental

a microfluorometer

bacterial

re-

amount of background to cellular components

consisting

for

to

major

potential

been

making

which

fluorescence

between data quinacrine-DNA has

experimental laser

The low

is another

scattering

species

A small bound

comparisons data on soluble of

state

the

or chromosomes. from quinacrine

of

material. Time for investigating

involved with biologic often contain multiple

resolve.

encountered

feasibility

cytologic advantages

excited

single cells fluorescence, quantitative tions and

the

analyses on has several

or

heterogeneous

and other excited sequence-specific

DISCUSSION

of

found extensive quenching of quinacrine fluorescence by satelite DNA having two dGC base pairs per seven nucleotide repeating sequence whereas one dGC pair did not have this effect.

component to A-T-rich

(4,

alternating

Ib).

in

and

upon

to marked

for DNA of different Rigler (20) have shown

(Table

on in

DNA.

little

and suggested are proportional

produced

that

found

yields)

as seen

increase

the

those

dependent

of fluorescence the intercalation

of the

with

d(TG-AC)

with

findings

moderate

such

solutions and we observed

quantum

leads

exhibits

accounting

fluorescence pairs. This

a

features

of complexes

prises 40% of the DNA of the organism and another satellite with 65% A-T contributes a further 13% of the genome (17). The decay curves measured for the S. leonensis nuclei showed a marked increase in the contribution from the longer

The

to

predomwith one

limited

of polynucleotides

bases

other

enhancement at or near

contains three two and two for

(and

purine

with

parent affinity Pachmann and

mately half of the total DNA of diploid cells, and nuclei of these cells exhibit foci of moderately bright quinacrine fluorescence. The latter organism shows a large region in inter-

surements.

constituent

Quinacrine

lifetimes

light

strikingly

of deoxyguanylate

marked interactions

was

are consistent on a variety

The emission intensities crine-DNA complexes are

presence

to

(15)

comparable intensity both in not be measured. The lifetimes

microscope preparations solution studies conducted naturally occurring DNA.

are

composition

dependence

preparations

of quinacrine-stained

phase from

of could

structural the

fluorescence

G-C pair

a similar

in a microscope

samples slides

photomulti-

increments

components quinacrine

attempted

curve

wavelength-de-

of the

of quinacrine lymphocytes

of the

response

The

(transit

for by varying

of the two Y chromosome

to be decay

midpoint

contribution.

records by one fitted parameters.

fluorescence male and

intensities creased

the

response

corrected

and sample mixed the

at

scatter

AL.

are samples

very as

FLUORESCENCE

seen

in Figure

4. In

addition,

A-T satellite the predominance

to the

for

fce

T)]

which

the

primary

is 75%,

probably

and the

DNA

de

(leading

bands,

which

A-T

(leading

(to the In

the

The

highly

relatively

Austin,

We thank

Texas.

the

manuscript. and a Research National Institute

G: Laser

A, Sacchi

CA,

microfluorometer

Svelto

Submitted

on

DD:

quinregions

in

in flow

by M Melamed, York, 1979, in press Latt SA: Optical studies

Rev

DNAs

of Dro-

Biol 38:417, 1973 10: Biophysical

Brodie

systems, PF

Flow

Mullaney,

Cytometry

and

M Mendelsohn.

Sorting.

Wiley,

of metaphase chromosome 5:1, 1976 probes of chromosome structure Cytol 19:603, 1977

Biophys

New

organization.

Bioeng

Latt SA: Fluorescent cation. Can J Genet

49:17, 15.

Satellite

Quant Walker

of quinacrine staining of chromosomes. 1974 KG: Hoechst 33258 fluorescent staining of Drosophila Chromosoma 49:333, 1975 Fluorescence polarization and energy transfer: theory

TM:

SA,

The

Symp GK,

mechanism

application

quinacrine rescence

Grinvald

for critical

reading

(GM21 from

17.

5, Munroe

with DNA of cytological

SR:

Optical

and chromatin: chromosome

studies

and

repli-

of complexes

implications preparations.

of

for the fluoChromosoma

1974

Loeser

CHN,

Clark

E,

Longoni

18.

Maher

M,

Tarkmeel

H:

Measurement

of

decay

G, Prenna

measurements

Electro Optics, 1977 A, Sacchi CA, Svelto 0: Fluoresmustard with DNA. I. Influence on the decay time in bacteria.

Zech L, Modest EJ, Foley GE, Wagh U, Simonsson fluorochromes for the study of the organization nucleus. Exptl Cell Res 58:141, 1969

4. Comings DE, Drets ME: Mechanisms of chromosome IX. Are variations in DNA base composition adequate

banding. to account

for quinacrine, Hoechst 33258 and daunomycin banding? Chromosoma 56:199, 1976 5. Duportail G, Mauss Y, Chambron J: Quantum yields and fluorescence lifetimes of acridine derivatives interacting with DNA. Biopolymers 16:1397, 1977 6. Dutrillaux B: New chromosome techniques, Molecular Structure of Human Chromosomes. Edited by JJ Yunis. Academic Press,

13:1038, 1974 JR: The organization

in drosophiidae.

Muller

W,

Chromosoma

Crothers

DM:

52:37,

Interaction

of

of interphase 1975 heterochromic

1972 of

parchrocom-

pounds with nucleic acids. I. The influence of heteroatoms and polarizabiity on the sequence specificity of intercalating ligands. Eur J Biochem 54:267, 1975

121) the 19.

A, Bottiroli

tides. Biochemistry Mayfield JE, Ellison

matin

20.

Muller chromic

W, B#{252}nemann H, compounds with

Dettagupta N: Interaction nucleic acids. II. Influence

chromic substituents intercalating ligands.

on the base Eur J Biochem

Pachmann

R:

U,

Rigler

Quantum

of heteroof hetero-

and sequence 54:279, 1975 yield

specificity

of acridines

of

interacting

with DNA of defined base sequence. Exptl Cell Res 72:602, 1972 21. Tsou KC, Giles B, Kahn G: On the chemical basis of chromosome banding patterns. Stain Technol 50:293, 1975 22.

for publication

3. Caspersson T, E: DNA-binding of the metaphase

Atherton

chromo-

fluorescence decay time in living cells. Exptl Cell Res 72:480, 16. Massari 5, Dell’Antone P, Colonna R, Azzone GF: Mechanism atebrin fluorescence changes in energized submitochondrial

for fluorescence

in biology. 3rd European Conf 2. Bottiroli G, Prenna G, Andreoni cence of complexes of quinacrine of the DNA base composition

Jovin

Annu

CITED 0,

EH,

of specific

13:2937,

Holmquist

14. Latt

or clustered

by a Research Grant Award (GM00122) Sciences.

LITERATURE 1. Andreoni

i.

13.

clustered.

Dr. Amiram

S. L. is supported Career Development of General Medical

on the

chromosomes. 1

12.

We wish to thank Dr. John Ellison, University of Texas, Austin, and Dr. Gerald Holmquist, Baylor University, Houston, for the preparation of S. leonensis and D. virilis slides, respectively. The former was obtained from the National Drosophila Species Resource Center of

Cohen

Edited

ACKNOWLEDGMENTS

at

10.

in

fluorescent

JG,

Biochemistry

probably Weisblum

extended

Gall

and

fluorescence

quenching)

represent

8.

studies

Almust it is

and

the

York, 1977, p 233-266 JR, Barr HJ: Quinacrine fluorescence some regions. Chromosoma 36:375, 1972

microscope

with

that

New

sophila virilis. Cold Spring Harb 9. Gottesfeld JM, Bonner J, Radda

A-T content will depend critically of dG residues, i.e., whether they

could are

form

agreement

conclude

quenching). pairs

fluorescence.

histones

to maximal

therefore, base

sequence

quinacrine

well.

we

to minimal

acrine

III). and

of

as

(25)

interspersed

Ia and content

fixation

chromosome regions of high on the statistical distribution are

(see Tables nucleotide

most

structure

Haseth

poly[d(A-

structure of the chromatin banding phenomenon,

since

removes

changes

for

95

QUINACRINE-DNA

7. Ellison

in time

to that

of

OF

of the

is reflected long decay

similar

tertiary quinacrine

secondary

specimens)

and

studies that the

ANALYSIS

contribution

fluorescence with the

a value

determinants

though proteins play a role in

enhanced

S. leonensis of the component

itself in solution Our data suggest

are

the

DECAY

Valeur by the

pulse 23. Valeur

24. 25.

B: Analysis of time-dependent method of modulating functions fluorometry. Chem Phys 30:85,

B, Moirez

J: Analyse

fluorescence with special 1978

des courbes

de decroissance

to

multiex-

ponentielles par la methode des functions modulatrices-application a la fluorescence. J Chim Phys 70:500, 1973 Weisblum B: Why centric regions of quinacrine-treated mouse chromosomes show diminished fluorescence. Nature 246: 150, 1973 Weisblum B, de Haseth PL: Quinacrine, a chromosome stain

specific for deoxyadenylate-deoxyathymidylate-rich DNA. Proc Nati Acad Sci USA 69:629, 1974 26.

experiments attention

Yguerabide

molecules, SN

Timasheff.

J:

Nanosecond

Methods

Academic

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fluorescence

in Enzymology, Press,

New

regions

spectroscopy

XXVI.

Edited

York,

1973,

in

of macro-

by CHW p 498-578

Him,