electronic supplementary material - genetics · 12/18/2003 · 068 0 69 genotypes and markers of...

Electronic Supplementary

Material


Material

Figure S1


Material

Figure S2


Material

Figure S3


Material

Table S1

Table

S1.

Olig

onucl

eotid

es

use

d in t

his

stu

dy

Nam

eS

equence

OD

LA

M131

5’-

CC

AG

GA

GA

CG

CA

AA

GT

TC

-3’

OD

LA

M137

5’-

TG

CT

TG

AA

TT

GT

CC

AT

AC

-3’

OD

LA

M138

5’-

GA

CC

CT

AC

GA

AC

CT

AA

GC

-3’

OD

LA

M141

5’-

CC

CA

AT

AC

AC

AT

AA

GA

CA

-3’

OD

LA

M149

5’-

GC

TT

GG

GG

AA

AA

TA

AT

CA

-3’

OD

LA

M157

5’-

TT

AC

CG

AC

TT

TC

TT

TA

CC

-3’

OD

LA

M158

5’-

AC

TA

TA

GT

CA

GC

GA

GG

AG

-3’

OD

LA

M159

5’-G

TC

GA

AT

TC

TC

AA

GC

CC

AT

CA

AT

CA

TC

AA

TC

GG

TC

AA

-3’

OD

LA

M160

5’-

AA

GG

GA

TC

CT

GG

AG

GC

GC

GA

TT

TA

GG

GG

TG

CT

GT

TA

C-3

’Q

DE

-2LD

5’-

AG

GG

GA

TC

CT

GC

CG

TT

CT

TG

TC

TC

CC

CT

GT

C-3

’Q

DE

-2LU

5’-

AG

GG

GA

TC

CC

CC

GG

TC

AG

GC

TC

GC

TC

CA

AC

T-3

’R

3IID

5’-

AG

GG

GA

TC

CC

GC

AT

CA

TA

TC

GC

CT

GT

CA

AA

T-3

’R

3IIU

5’-

AG

GG

GA

TC

CC

AA

CC

GG

CA

GA

AG

AA

GG

AA

GA

A-3

’


Material

Table S2

Tab

le S

2. M

arke

rs p

artic

ipat

ing

in a

sm

s-2+

/Sm

s-2R

IP2

cros

s sh

ow M

ende

lian

segr

egat

ion

Cro

ss A

Cro

ss B

Str

ains

:M

MN

CR

02A

x K

YN

CT

35A

MM

NC

R03

A x

KY

NC

T34

A

Mar

kers

:(h

is-3

; inl

; Sm

s-2R

IP2

A)

x(h

is-3

+::A

sm-1

+[3

430-

9336

]; in

l a)

(his

-3; i

nl; S

ms-

2RIP

2 a)

x(h

is-3

+::A

sm-1

+[3

430-

9336

]; in

l A)

Seg

rega

tion

mat

Am

at a

mat

Am

at a

Mat

ing

Typ

e35

3330

39

his-

3+hi

s-3

his-

3+hi

s-3

His

tidin

e A

uxot

roph

y34

3425

44

inl+

inl

inl+

inl

Inos

itol A

uxot

roph

y

068

069

Gen

otyp

es a

nd m

arke

rs o

f the

str

ains

use

d in

thes

e cr

osse

s ar

e de

scrib

ed in

Tab

le 2

. Cro

sses

and

the

anal

ysis

of t

he p

roge

ny w

ere

perf

orm

ed a

s de

scrib

ed in

Met

hods

(E

lect

roni

c S

uppl

emen

tary

Mat

eria

l). W

e an

alyz

ed a

tota

l of 6

8 an

d 69

rec

ombi

nant

s fr

om c

ross

esA

and

B, r

espe

ctiv

ely.


Material

Table S3

Table

S3. P

rote

ins

use

d in

the a

lignm

ent

Pro

tein

Org

anis

m1

Identif

ier

PP

DA

A. fu

mig

atu

s2

Contig

720

PP

DB

A. fu

mig

atu

s2

Contig

196

AG

O2

A. th

alia

na

AA

F24585.1

AG

O3

A. th

alia

na

AA

F24586.1

PIN

HE

AD

A. th

alia

na

AA

D40098.1

AG

O5

A. th

alia

na

AA

D21514.1

AG

O1

A. th

alia

na

AA

C18440.1

AG

O4

A. th

alia

na

AA

C77862.1

AG

O6

A. th

alia

na

AA

B91987.1

QD

E-2

B. gra

min

isA

AL06079.1

RD

E-1

C.

ele

gans

AA

F06159.1

ALG

-1C

. ele

gans

NP

_510322.1

T23D

8.7

C.

ele

gans

NP

_492643.1

T22B

3.2

aC

. ele

gans

T23164

PR

G-1

C.

ele

gans

NP

_492121.1

AG

O1

D.

mela

nogast

er

BA

A88078.1

AU

BD

. m

ela

nogast

er

AA

F53046.1

PIW

ID

. m

ela

nogast

er

AA

F53043.1

AA

L57170.1

D.

rerio

AA

L57170.1

BA

B13393.1

H. sa

pie

ns

BA

B13393.1

AA

H28581.1

H. sa

pie

ns

AA

H28581.1

QD

E-2

N.

crass

aA

AF

43641.1

SM

S-2

N.

crass

aN

CU

09434.1

eIF

2c

O.

cunic

ulu

sA

AC

24323.1

PA

PP

. ca

udatu

mB

AA

88525.1

T41568

S. pom

be

T41568

AA

G42533.1

S. purp

ura

tus

AA

G42533.1

1G

eneB

ank

acc

ess

ion n

um

bers

. 2

Unfin

ished A

sperg

illus

fum

igatu

s G

enom

e P

roje

ct a

t: h

ttp://w

ww

.tig

r.org

(T

he Inst

itute

for

Genom

icR

ese

arc

h).


Methods

Lee, Dong W., Pratt, Robert J., McLaughlin, Malcolm and Aramayo, RodolfoPage 1

Methods

Procedures for DNA extraction from Neurospora crassa, Southern blot analysis, and

other nucleic acid manipulations were performed as described (Pratt and Aramayo

2002). Similarly, growth conditions, conidial spheroplast preparation, fungal

transformation, homokaryon purification, female fertility/sterility determinations, and

genetic crosses were performed as described (Pratt and Aramayo 2002). The formulas

for the Vogel’s Medium N, the Westergaard’s Medium, and the sugar mixture of

Brockman and de Serres have been described by Davis and de Serres (1970).

Strain Description. Strains of N. crassa are described in Table 2. Escherichia coli K12

XL1-Blue MR (Stratagene, La Jolla, CA, USA) was the host for all our bacterial

manipulations. When non-methylated DNA was needed for enzyme digestions, either

GM2163--an E. coli K12 derivative containing, among others markers, a dam13::Tn9

(CamR) and a dcm-6 mutations (New England BioLabs (NEB), Beverly, MA, USA), or

JM110--an E. coli K12 derivative containing, among others, dam and dcm mutations

(Yanisch-Perron et al. 1985), was used.

A Note About the Integration at his-3 Locus. Due to the nature of the his-3

integration vectors used in this study (Aramayo and Metzenberg 1996), the

lysophospholipase, (lpl) gene, located downstream of the his-3 gene was deleted during

the integration of our constructs. Arbitrarily defining the HindIII restriction site present in

the coding region of his-3 as position 1, the region deleted during the gene replacement


spans from position 5192 to position 6046. We include this deletion (lpl∆(5192-6046)) as

part of the genotype of all our strains containing integrations at the his-3 locus (Table 2),

to distinguish them from strains containing integrations at the his-3 locus obtained with a

third generation of his-3-integration vectors (Margolin et al. 1997). The genome

sequence of the histidine-3 (his-3+) locus can be found on contig 3.165 (Release 3,

Whitehead Institute--http://www-genome.wi.mit.edu/annotation/fungi/neurospora/).

A Note About Strains Containing the Asm-1∆∆∆∆(3430-9336)::hph+::mcl-1 Deletion

Allele of Asm-1. In these strains, the region deleted encompassed a region predicted

to direct the transcription of a gene that we call myosin chain-like-1 (mcl-1+, Figure 1),

based on its weak homology to myosin-chain-like genes in other organisms (data not

shown). Strains containing a disruption in this predicted gene are viable and do not

have any detectable developmental or metabolic phenotypes.

DNA Sequencing. The Sad-1RIP64 allele (Suppressor of ascus dominance-1 [RIP64])

was sequenced using oligonucleotides ODLAM131, ODLAM137, ODLAM138,

ODLAM141, ODLAM149, ODLAM157, and ODLAM158 (Table S1) as primers, from a

2,711 bp PCR-amplified fragment (coordinates 1239 to 3950) from DLNCR64A

chromosomal DNA using oligonucleotides R3IIU and R3IID (Table S1). We used the

BigDye™ Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA

polymerase (PEBiosystems, Foster City, CA, USA). Sequencing of Sad-1RIP64

revealed, among the many GC to AT transition mutations typical of RIP (see Electronic

Supplementary Material, Figure S1), an ATG to an ATA mutation at the translation-start


codon. Thus no SAD-1 polypeptide should be synthesized by this allele. The GenBank

Accession Number corresponding to the Sad-1RIP64 allele is AF500110.

Plasmids pDLAM102 (suppressor of meiotic silencing-2--Sms-2RIP2 allele) and

pDLAM103 (Sms-2RIP88 allele) were sequenced using both the GeneJumper Primer

Insertion Kit (Invitrogen, Carlsbad, CA, USA) and the BigDye™ Terminator Cycle

Sequencing Ready Reaction Kit with AmpliTaq DNA polymerase (PEBiosystems). The

GenBank Accession Numbers corresponding to the sms-2+, Sms-2RIP2 and Sms-

2RIP88 alleles are AF508210, AF508211 and AF508212, respectively.

Sequences were generated on an Applied Biosystems Model 377 or 373 automated

DNA sequencer at GeneTechnologies Laboratory (Institute of Developmental and

Molecular Biology—IDMB, Texas A&M University, College Station, TX, USA).

Plasmid Construction. The Sad-1 locus is contained on contig 3.102 (Release 3,

Whitehead Institute--http://www-genome.wi.mit.edu/annotation/fungi/neurospora/). We

arbitrarily defined as position 1 the SphI site located 2,031 bp upstream of the

translational initiation signal (ATG) for SAD-1 (NCU02178.1).

The genome sequence of the Ascospore maturation-1 (Asm-1) locus is contained on

contig 3.56 (Release 3, Whitehead Institute--http://www-

genome.wi.mit.edu/annotation/fungi/neurospora/). We arbitrarily defined as position 1

the HindIII site located 6,126 bp upstream of the translational initiation signal (ATG) for


ASM-1 (Aramayo et al. 1996). Following this convention the HindIII fragment contained

in pRAUW44 (Aramayo et al. 1996) maps from coordinates 1 to 12425.

The genome sequence of the Sms-2 locus is contained on contig 3.602 (Release

3, Whitehead Institute--http://www-genome.wi.mit.edu/annotation/fungi/neurospora/).

We arbitrarily defined as position 1 the sequence corresponding to the 5’-most base

located downstream of the EcoRI site present in the ODLAM159 oligonucleotide

(coordinates 5507 to 5534, Table S1). Position 1 is located 1,951 bp upstream of the

translational initiation signal (ATG) for SMS-2 (NCU09434.1).

Plasmids pOKE76 and pNNAid both were generously provided by Robert L.

Metzenberg. Plasmid pOKE76 consists of the 3,363 bp PvuII-SphI fragment from the

mat a-1 idiomorph of N. crassa, cloned into the HincII-SphI sites of pGEM3Zf(+)

(Promega, Madison, WI, USA), whereas plasmid pNNAid consists of the 5,758 bp NdeI-

NsiI fragment of the mat A-1 idiomorph inserted into the NdeI-NsiI sites of pGEM5Zf(-)

(Promega).

The following plasmids were constructed following standard procedures (Ausubel et al.

1987; Sambrook et al. 1989):

pDLAM092. Constructed by inserting the 5,534 bp PCR product amplified from

RANCR06A chromosomal DNA using ODLAM159 (coordinates 5507 to 5534) and

ODLAM160 (coordinates 1 to 28, Table S1) as primers into EcoRI-BamHI sites of


pRAUW122 (Aramayo and Metzenberg 1996). The PCR product was digested with

EcoRI and BamHI prior to ligation.

pDLAM102. Constructed by ligating the 5,534 bp PCR product amplified from

MMNCR03A chromosomal DNA using ODLAM159 (coordinates 5507 to 5534) and


pBluescript II SK(+) (Stratagene). The PCR product was digested with EcoRI and

BamHI prior to ligation.

pDLAM103. Constructed by ligating the 5,534 bp PCR product amplified from

DLNCR88A chromosomal DNA using ODLAM159 (coordinates 5507 to 5534) and


pBluescript II SK(+) (Stratagene). The PCR product was digested with EcoRI and

BamHI prior to ligation.

pKYAM001. Constructed by cloning the 2,472 bp EcoRI-KpnI fragment (coordinates

958 to 3430) from pRAUW63 (Aramayo et al. 1996) into the EcoRI-KpnI sites of

pGEM3Zf(+) (Promega).

pKYAM002. Constructed by cloning the 1.4 kb HpaI fragment containing the hph+ gene

from pCB1004 (Carroll et al. 1994) into the SmaI site of pKYAM001.

pKYAM003. Constructed by cloning the 3,089 bp XbaI-HindIII fragment (coordinates

9336 to 12425) from pRAUW44 (Aramayo et al. 1996) into the XbaI-HindIII sites of

pKYAM002.

pKYAM006. Constructed by cloning the 1,241 bp KpnI fragment (coordinates 3430 to

4671) from pRAUW44 (Aramayo et al. 1996) into the KpnI site of pBCKS(+)


(Stratagene). asm-1+ and lacZ+ have the opposite direction of transcription in this

plasmid.

pKYAM005. Constructed by cloning the 4,727 bp EcoRI-XbaI fragment (coordinates

4615 to 9336) from pRAUW44 (Aramayo et al. 1996) into the EcoRI-XbaI sites of

pRAUW122 (Aramayo and Metzenberg 1996).

pKYAM011. Constructed by cloning the 1,242 bp EcoRI fragment from pKYAM006 into

the EcoRI site of pKYAM005.

pKYAM052. Constructed by cloning the 3,610 bp EcoRI fragment containing the mat a-

1+ idiomorph of N. crassa from pOKE76 into the EcoRI site of pKYAM003.

pKYAM055. Constructed by cloning the 1,687 bp ScaI-EcoRI fragment from pNNAid,

containing the mat A-1+ gene of N. crassa into the HindIII site of pKYAM003. The ScaI-

EcoRI fragment from pNNAid (see above) and the HindIII fragment from pKYAM003

were both treated with Klenow polymerase (NEB) in the presence of dNTPs prior to

their ligation.

pQde-2l. Constructed by inserting a 2,374 bp PCR product amplified from RANCR06A

DNA using QDE-2LU (coordinates 4447 to 4468) and QDE-2LD (coordinates 2095 to

2116, Table S1) as primers into BamHI site of pRAUW122 (Aramayo and Metzenberg

1996). The PCR product was digested with BamHI prior to ligation.

pRdRP3. Constructed by cloning the 2,711 bp PCR-amplified fragment (coordinates

1239 to 3950, GeneBank AF500110) from wild-type genomic DNA (RANCR06A) using

R3IIU and R3IID as primers (Table S1), into the BamHI site of pRAUW122 (Aramayo

and Metzenberg 1996). The PCR product was digested with BamHI prior to ligation.


Bioinformatics of SMS-2. SMS-2 homologs listed in Table S3 were aligned with T-

Coffee v1.37 using the fast_pair method and the Vasiliky simulation matrix (Notredame

et al. 2000). The poorly aligned N-terminal region was removed and the sequences

unaligned. The trimmed sequences were then aligned with DiAlign2

(http://www.genomatix.de/cgi-bin/dialign/dialign.pl) (Morgenstern et al. 1996;

Morgenstern et al. 1998), ClustalX (using the Gosset series matrix) (Jeanmougin et al.

1998), PIMA (using the maximal linkage and sequential branching cluster methods;

http://searchlauncher.bcm.tmc.edu:9331/multi-align/multi-align.html) (Smith and Smith

1990; Smith and Smith 1992), and the slow_pair method of T-Coffee v1.39 (using the

Vasiliky simulation matrix) (Notredame et al. 2000). T-Coffee v1.39 was then used to

create a consensus alignment from these five alignments. The consensus alignment

was manually edited. Bayesian, or most posterior probability trees were generated with

MrBayes v2.01 (Huelsenbeck and Ronquist 2001; Huelsenbeck et al. 2001) using the

“Jones” substitution model, 100,000 generations, building an “all compatible” type tree

consensus and “burning” (ignoring) the trees from the first 10,000 generations with

sampling every 10 generations. Protein domains were identified by submitting SMS-2 to

the SMART database (http://smart.embl-heidelberg.de/) (Letunic et al. 2002; Schultz et

al. 1998) and the Pfam database (Bateman et al. 2002; Bateman et al. 2000;

Sonnhammer et al. 1997) by clicking this option at the SMART website.


References.

Aramayo, R., and R. L. Metzenberg, 1996 Gene replacements at the his-3 locus of

Neurospora crassa. Fungal Genetics Newsletter 43: 9-13.

Aramayo, R., Y. Peleg, R. Addison and R. Metzenberg, 1996 Asm-1+, a Neurospora

crassa gene related to transcriptional regulators of fungal development. Genetics

144: 991-1003.

Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith et al. (Editors), 1987

Current Protocols in Molecular Biology. John Wiley and Sons, New York.

Bateman, A., E. Birney, L. Cerruti, R. Durbin, L. Etwiller et al., 2002 The Pfam protein

families database. Nucleic Acids Research 30: 276-280.

Bateman, A., E. Birney, R. Durbin, S. R. Eddy, K. L. Howe et al., 2000 The Pfam protein

families database. Nucleic Acids Research 28: 263-266.

Carroll, A. M., J. A. Sweigard and V. Valent, 1994 Improved vectors for selecting

resistance to hygromycin. Fungal Genetics Newsletter 41: 22.

Davis, R. H., and F. J. de Serres, 1970 Genetic and microbiological research

techniques for Neurospora crassa, pp. 79-143 in Metabolism of Amino Acids and

Amines, edited by S. P. Colowick and N. O. Kaplan. Academic Press, New York

and London.

Huelsenbeck, J. P., and F. Ronquist, 2001 MRBAYES: Bayesian inference of

phylogenetic trees. Bioinformatics 17: 754-755.


Huelsenbeck, J. P., F. Ronquist, R. Nielsen and J. P. Bollback, 2001 Bayesian

inference of phylogeny and its impact on evolutionary biology. Science 294:

2310-2314.

Jeanmougin, F., J. D. Thompson, M. Gouy, D. G. Higgins and T. J. Gibson, 1998

Multiple sequence alignment with Clustal X. Trends in Biochemical Sciences 23:

403-405.

Letunic, I., L. Goodstadt, N. J. Dickens, T. Doerks, J. Schultz et al., 2002 Recent

improvements to the SMART domain-based sequence annotation resource.

Nucleic Acids Research 30: 242-244.

Margolin, B. S., M. Freitag and E. U. Selker, 1997 Improved plasmids for gene targeting

at the his-3 locus of Neurospora crassa by electroporation. Fungal Genetics

Newsletter 44: 34-36.

Morgenstern, B., A. Dress and T. Werner, 1996 Multiple DNA and protein sequence

alignment based on segment-to-segment comparison. Proceedings of the

National Academy of Sciences of the United States of America 93: 12098-12103.

Morgenstern, B., K. Frech, A. Dress and T. Werner, 1998 DIALIGN: finding local

similarities by multiple sequence alignment. Bioinformatics 14: 290-294.

Notredame, C., D. G. Higgins and J. Heringa, 2000 T-Coffee: A novel method for fast

and accurate multiple sequence alignment. Journal of Molecular Biology 302:

205-217.

Pratt, R. J., and R. Aramayo, 2002 Improving the efficiency of gene replacements in

Neurospora crassa: a first step towards a large-scale functional genomics

project. Fungal Genetics and Biology 37: 56-71.


Sambrook, J., E. F. Fritsch and T. Maniatis (Editors), 1989 Molecular cloning: a

laboratory manual. Cold Spring Harbor Laboratory Press, N. Y.

Schultz, J., F. Milpetz, P. Bork and C. P. Ponting, 1998 SMART, a simple modular

architecture research tool: identification of signaling domains. Proceedings of the

National Academy of Sciences of the United States of America 95: 5857-5864.

Smith, R. F., and T. F. Smith, 1990 Automatic generation of primary sequence patterns

from sets of related protein sequences. Proceedings of the National Academy of

Sciences of the United States of America 87: 118-122.

Smith, R. F., and T. F. Smith, 1992 Pattern-induced multi-sequence alignment (PIMA)

algorithm employing secondary structure-dependent gap penalties for use in

comparative protein modelling. Protein Engineering 5: 35-41.

Sonnhammer, E. L., S. R. Eddy and R. Durbin, 1997 Pfam: a comprehensive database

of protein domain families based on seed alignments. Proteins 28: 405-420.

Yanisch-Perron, C., J. Vieira and J. Messing, 1985 Improved M13 phage cloning

vectors and host strains: nucleotide sequences of the M13mp18 and pUC19

vectors. Gene 33: 103-119.

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