Download Sequence of Actin cDNA from Fucus disticus`

Plant Physiol. (1995) 107: 1007-1 008
Plant Gene Register
Sequence of Actin cDNA from Fucus disticus'
Bradley W. Coodner2, Janice D. Davis, and Ralph S. Quatrano*
Biology Department, University of North Carolina, Chapel Hill, North Carolina 27599
The brown alga Fucus disticus is an appropriate model
system for the study of polarity establishment during embryogenesis (Goodner and Quatrano, 1993). The actin cytoskeleton is required for the formation and fixation of an
axis of polarity (Quatrano, 1973) and becomes localized to
the site of future rhizoid outgrowth during axis fixation
(Kropf et al., 1989).Actin mRNA is stored in the egg and is
translated throughout embryogenesis, with the steadystate level of actin protein remaining constant (Kropf et al.,
1989; Masters et al., 1992). However, there are three isoforms of actin protein present in Fucus zygotes, and it is not
known whether these isoforms perform different functions
(Kropf et al., 1989).
As part of a larger project to identify genes involved in
Fucus polarity establishment, we isolated and characterized actin cDNA clones (Table I). An interna1 fragment of
an actin gene was amplified by PCR and used to identify
c l o n e s from Fucus e m b r y o and vegetative frond cDNA
libraries. Restriction endonuclease mapping of a11 of the
positive cDNA clones from each library and sequencing
of severa1 clones suggest that there is only one expressed
actin gene. There appear to be two major polyadenylation sites, 593 and 768 nucleotides downstream of the
last codon. The significance of these two polyadenylation sites is currently unknown. Southern blot analysis of
genomic DNA confirmed that there is only one gene
encoding actin in the F . disticus genome. Therefore, the
three actin isoforms present in embryos are the products
of posttranslational modification of a single actin protein. Comparison of the encoded polypeptide sequence
with actins from other organisms supports the close
phylogenetic relationship between brown algae and oomycetes (Bhattacharya et al., 1991; Baldauf and Palmer,
1993).
The cDNA reported here has been used in in situ hybridizations to determine the spatial distribution of actin
mRNA within developing Fucus zygotes and embryos
(F.-Y. Bouget, S. Gerttula, and R.S. Quatrano, unpublished
data).
'This research was supported by grants t o R.S.Q. f r o m the
National Science Foundation (No. M C B 9318757) and the Office of
N a v a l Research (No. N00014-91-J-4128).
Present address: Department of Biology,
-_ University of Richmond, Richmond, VA 23173.
* Corresponding author; e-mail [email protected]; fax
1-91 9 -962- 0778.
Table 1. Characteristics o f actin cDNA from Fucus disticus
Organism:
Fucus disticus; eggs and sperm were shed from reproductive
fronds collected near Hatfield Marine Science Center, Newport, OR.
Gene Location:
Single copy, nuclear gene.
Gene Product:
Actin; subunit of microfilament cytoskeleton.
Source:
Embryo c D N A library in A-ZipLox constructed using poly(A)+
RNA isolated from 24-h-old embryos; a gift from Crispin Taylor (University of North Carolina, Chapel Hill). Vegetative
cDNA library in A-ZAPII constructed using poly(A)+ RNA isolated from vegetative and reproductive frond tips; a gift from
Lynda Goff (University of California at Santa Cruz).
Method of Identification:
An actin gene fragment was amplified by PCR using genomic
D N A from Ascophyllum, a related brown alga, as template,
and degenerate primers based on conserved regions of actin
protein sequence. Primers were a gift from John McDowell
and Richard Meagher (University of Georgia, Athens). The actin gene fragment was used as a probe to screen Fucus embryo and vegetative cDNA libraries.
Sequencing:
Double-stranded plasmid sequencing of both strands by the
dideoxynucleotide chain termination method.
Features of cDNA:
The largest actin cDNA consists of a 45-nucleotide-long 5' untranslated region, a 1 125-nucleotide-long open reading frame,
and a 768-nucleotide-long 3 ' untranslated region.
Codon Usage:
33% G, 6% A, 14% T, and 47% C in the third position.
Features of Protein:
Open reading frame encodes a polypeptide of 375 amino acid
residues. Sequence identity on the amino acid level with selected actins from other organisms is as follows: Costaria
costata (Bhattacharya et al., 1991), 98%; Achlya bisexualis
(Bhattacharya et al., 1991), 93%; Phytophthora megasperma
(Dudler, 1990), 84%; maize M A c l (Shah et al., 19831, 80%;
Saccharomvces cerevisiae (Gallwitz and Sures, 1980). 80%.
Received August 11, 1994; accepted August 29, 1994.
Copyright Clearance Center: 0032-0889/95 /107/ 1007/02.
The GenBank accession number for the sequence reported in this
article i s U11697.
LITERATURE ClTED
Baldauf SL, Palmer JD (1993) Animals a n d fungi are each other's
closest relatives: congruent evidence f r o m multiple proteins.
Proc N a t l Acad Sci USA 9 0 11558-11562
1007
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1008
Goodner et al.
Bhattacharya D, Stickel SK, Sogin ML (1991) Molecular phylogenetic analysis of actin genic regions from Achyla bisexualis (Oomycota) and Costaria costata (Chromophyta). J Mo1 Evol 3 3 525-536
Dudler R (1990) The single copy actin gene of Phytophthora megasperma. Plant Mo1 Biol 14: 415-422
Gallwitz D, Sures I (1980)Structure of a split yeast gene: complete
nucleotide sequence of the actin gene in Saccharomyces cerevisiae.
Proc Natl Acad Sci USA 77: 2546-2550
Goodner B, Quatrano RS (1993) Fucus embryogenesis: a model to
study the establishment of polarity. Plant Cell 5 1471-1481
Plant Physiol. Vol. 107, 1995
Kropf DL, Berge SK, Quatrano RS (1989) Actin localization during Fucus embryogenesis. Plant Cell 1: 191-200
Masters AK, Shirras AD, Hetherington AM (1992) Maternal
mRNA and early development in Fucus serratu:. Plant J 2:
619-622
Quatrano RS (1973) Separation of processes associated with differentiation o f two-celled Fucus zygotes. Dev Biol 30: 209-213
Shah DM, Hightower RC, Meagher RB (1983) Genes encoding
actin in higher plants: intron positions are highly conserved but
the coding sequences are not. J Mo1 Appl Gen 2 111-126
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