cific habitats in natural populations of Arabidopsis.
From the Department of Biochemistry, 420 Henry Midi,
University of Wisconsin, Madison Wl 53706-1569. This
work was supported by a grant from the U.S. Department of Agriculture to R.MA (95-37100-1614) and by
the College of Agricultural and life Sciences, University
of Wisconsin, Madison. We thank K. Omata, A. Masshardt and J. Pomerenlng for assistance with this work.
Address correspondence to R. Amaslno at the address
above.
The Journal of Heredity 1997*8(1)
References
Bell CJ and Ecker JR, 1994. Assignment of 30 mlcrosatelllte loci to the linkage map of Arabidopsis. Genomlcs
19:137-144.
Bum JE, Smyth DR, Peacock WJ, and Dennis ES, 1993.
Genes conferring late flowering In Arabidopsis thaliana
Genetlca 90:147-155.
Clarke JH and Dean C, 1994. Mapping FRI, a locus controlling flowering time and vernalization response In
Ambidopsis thaliana. Mol Gen Genet 242:81-89.
Harer L, 1950. Die Vererbung des Bluhalters fruher und
spater sommerelnjahriger Rassen von Arabidopsis thaliana (L) Heynh. Beltr Blol Pflanzen 28:1-35.
Karlovska V, 1974. Genotyplc control of the speed of
development In Arabidopsis thaliana (L.) Heynh. lines
obtained from natural populations Blol Plantarum 16:
107-117.
Koornneef M, Blankesti]n-de Vries H, Hanhart C, Soppe
W, and Peeters T, 1994. The phenotype of some late
flowering mutants Is enhanced by a locus on chromosome 5 that Is not effective In the Landsberg erecta
wild-type. Plant J 6:911-919.
Napp-ZInn K, 1979. On the genetlcal basis of vernalization requirement In Arabidopsis thaliana (L) Heynh.
In- La Physlologie de la Floraison (Champagnat P and
Jaques R, eds). Paris: Colloques Internatlonaux du Centre National de la Recherche Sdentlfique; 217-220.
Napp-ZInn K, 1985. Ambidopsis thaliana. In: CRC handbook of Dowering (Halevy AH, ed). Boca Raton, Florida:
CRC Press; 492-503.
Ratcllffe D, 1961. Adaptation to habitat in a group of
annual plants. J Ecol 49:187-203.
Vince-Pnie D, 1983. Photomorphogenesls and flowerIng. In: Encyclopedia of plant physiology, New Series:
Photomorphogenesls (Shropshire W and Mohr H, eds).
Berlin: Sprlnger-Verlag; 457^90.
Received August 7, 1995
Accepted March 11, 1996
Corresponding Editor Prem P. Jauhar
Molecular and Cytologlcal
Analysis of a Mariner
Transposon From
Hessian Fly
V. W. Russell and R. H. Shukle
Degenerate PCR primers for conserved
regions of the mariner transposase have
been shown to amplify DNA sequences
from the Hessian fly (Mayetiola destructor). Using one of these sequences as a
hybridization probe, a clone from an M.
destructor genomic library in phage lambKoomneef M, Hanhart CJ, and van der Veen JH, 1991.
A genetic and physiological analysis of late flowering
da was recovered and sequenced. A
mutants In Arabidopsis thaliana. Mol Gen Genet 229:57transposable element, Desmari, with per66.
fect inverted terminal repeats and an open
Kowalskl SP, Lan TH, Feldmann KA, and Paterson AH,
reading frame that encodes a mariner
1994. QTL mapping of naturally-occurring variation In
flowering time of Arabidopsis thaliana. Mol Gen Genet class transposase was found. When com245:548-555
pared to mariner sequences in the gene
Lalbach F, 1951. Uber sommer- und wlnterannuale Rasdatabase, the transposase proved to be
sen von Arabidopsis thaliana (L) Heynh. Eln Beltrag
similar to that of the active mariner Mos1
zur Atlologie der Blutenblldung. Beitr Blol Pflanzen 28:
73-210.
from the fruit fly (Drosophila mauritiana). In
situ hybridization of the transposon DNA
Lee I and Amaslno RM, 1995. Effect of vernalization,
photoperiod and light quality on the flowering phenosequence to salivary gland polytene chrotype of Arabidopsis plants containing the FRIGIDA gene.
mosomes revealed the general cytological
Plant Physlol 108.157-162.
locations of mariner elements. The distriLee I, Bleecker A, and Amaslno R, 1993. Analysis of naturally occurring late Dowering In Arabidopsis thaliana. bution of sequences with homology to the
Mol Gen Genet 237:171-176.
probe was predominantly, but not excluLee I, Michaels SD, Masshardt AS, and Amaslno RM, sively, in paracentromeric regions.
1994. The late flowering phenotype of FRIGIDA and LUMINIDEPENDENS Is suppressed in the Landsberg erecta
strain of Arabidopsis. Plant J 6:903-909.
Transposable elements are powerful reMartlnez-Zapater JM, Coupland G, Dean C, and Koornsearch tools for genetic manipulation and
neef M, 1994. The transition to flowering In Arabidopsis.
In: Arabidopsis (Meyerowltz E and SomerviUe CR, eds). investigation but such research has been
limited to only a few organisms. A search
Plalnvtew, New York: Cold Spring Harbor Laboratory
Press; 403-133.
for useful transposable elements has led
Michaels SD, John MJ, and Amaslno RM, 1994. Removal
to the discovery of numerous types of
of polysaccharides from plant DNA by ethanol precipthese elements across a wide phylogenic
itation. BloTechnlques 17:274-276.
range. One such element is mariner, a
Murashlge T and Skoog F, 1962. A revised medium for
short inverted repeat class, which transrapid growth and bioassay with tobacco tissue cultures. Physlol Plant 15:473-497.
poses via a DNA intermediate, and was
Nam H-G, Glraudat J, den Boer B, Moonan F, Loos WDB, first described in the fruit fly (Drosophila
Hauge BM, and Goodman HM, 1989. Restriction fragmauritiana) (Jacobson et al. 1986). The
ment length polymorphism linkage map of Arabidopsis
mariner element has since been detected
thaliana. Plant Cell 1:699-705.
7 2 The Journal of Heredity 1997.88(1)
in a wide variety of insects using PCR with
primers designed to conserved regions of
the transposase (Bigot et al. 1994; Robertson 1993; Robertson and MacLeod 1993).
Many of these mariner elements have been
shown to be defective, lacking a complete
transposase, and the role and specificity
of the Inverted terminal repeats is not
clear. To date only the mariner elements
from Drosophila have been shown to be
active, especially the Mosl mariner element from D. mauritiana, and have been
shown to be capable of transforming other
closely related Insects in the genus Drosophila (Maruyama et al. 1991). There is a
need to characterize other mariner elements in order to understand the potential of these elements for genetic studies.
We previously documented evidence of
mariner transposable elements from the
Hessian fly (Mayetiola destructor, family
Cecidomyiidae, order Diptera), a major
pest of wheat, using PCR to amplify fragments with degenerate primers designed
to conserved regions of the mariner transposase. One of these elements, Desmarl,
has now been sequenced and appears to
be in the subfamily of mariner elements
that was first characterized in D. mauritiana. This sequence, representing an intact
mariner element with perfect inverted repeats, and its Insertion site are presented
here. The distribution of this sequence in
the genome is described using in situ hybridization to salivary polytene chromosomes.
Materials and Methods
Experimental Insect
Hessian flies (white-eyed line; Shukle and
Stuart 1993) were reared on wheat cultivar
Blueboy (contains no known resistance
genes) in a growth chamber at 20°C with
a 16 h photoperiod (illumination 360 (xEin/
mVs).
Construction and Screening of Library
Hessian fly genomic DNA was prepared according to the method of Us et al. (1983).
A phage lambda library was constructed
from a partial Saul digest of Hessian fly
genomic DNA using the lambda BlueStar
Xhol half-site cloning system from Novagen (Madison, Wisconsin).
Desl, a 345 bp PCR product representing a conserved sequence of a mariner
transposase (Shukle and Russell 1995)
was labeled with [32P]dCTP (sp.act. 3,000
Ci/mmol; Amersham, Arlington Heights, Illinois) by ollgonucleotide random priming
reaction (Feinberg and Vogelstein 1983)
1211
934
629
168
regions of homology with the inverted repeats of Mosl (67% identity), including
nine continuous nucleotides (5'-GGTGTACAA). Similar homology can be seen in
comparisons with several other species
(Ebert et al. 1995; Jeyaprakash and Hoy
1995), and in several hymenopteran species there is also close homology at the
opposite end of the repeat (AAACCGGAATT-3') (Bigot et al. 1994) (Figure
2)Downstream from the inverted repeat at
the 5' end of the mariner is a region that
contains several presumptive promotor
CATA sites and is also characterized by
several sequences of homology to the region flanking the transposase of Mosl.
Most notable is a conserved region immediately downstream from the inverted
repeat, which appears in all of the mariners sequenced to date (Figure 3). Such
conservation should indicate an important recognition site for transposition.
Work on D. mauritiana indicates that
regions beyond the inverted repeat appear to be involved in site-specific DNA
binding protein recognition (Hartl DL, personal communication). Such a protein
binding site has been shown to be a characteristic of P-element transposition
(Kaufman et al. 1989).
An ATG at nucleoUde 168 initiates an intact open reading frame of 1,041 bp, which
terminates with a GAA (glutamic acid)
and TAA (ochre terminator) and which
codes for the putative transposase. When
compared to the family of mariner sequences deposited in Genbank, a family
that consists of 13 distinct subfamilies
(Robertson and Lampe 1995), Desmarl
proved to be in the D. mauritiana subfamily. A comparison with the Mosl mariner
element from D. mauritiana shows an
overall identity of 59% with Desmarl along
the whole element, while the inferred ami-
AAGTTGTACACCCAA
TTGGGTGTACAACTTAAAAACCGGAATT
Figure 1. Diagram of the mariner element Desmarl recovered from Hessian fly with Desl. The element was 1,288
bp in length with terminal perfect Inverted repeats of 28 bp (shown In light gray). An ATG at nucleoUde 168 Initiates
an open reading frame for a putative transposase of 1,041 bp (dark gray including the striped region) which
terminates with a GAA (glu) and TAA (ochre terminator). The sequence upstream from the Initiation site contains
several putative promotor elements: CAAT (123), CATA (150), and the highly conserved site for the selection of
the AUG Initiation codon; A at - 3 . The termination sequences GAATAA may serve as a polyadenylatlon signal
(Haiti 1989). The conserved region of the transposase used for PCR analysis of mariner elements In the Hessian
fly Is Indicated by stripes (629-934).
and was used to recover mariner sequences from the lambda library. A 3 kbp EcoRl
fragment from one of the lambda clones
was subcloned into the vector pGEM7Z
(Promega, Madison, Wisconsin) and sequenced enzymatically (Sanger et al.
1977) using internal primers to isolate the
mariner sequence.
The mariner element and approximately
150 bases on either side were sequenced
repeatedly on both strands. Sequence
comparisons were performed using BLAST
from Genbank.
typical of all mariner elements, and has
perfect inverted terminal repeats of 28 bp
(Figure 1). With the exception of the perfect inverted terminal repeat of the honeybee (Apis mellifora; Ebert et al. 1995), all
of the reported mariner elements have
proved to have imperfect repeats. Many of
these elements, including those of A. mellifora, have been found to contain numerous mutations, and most are considered
inactive. Inverted terminal repeats play a
fundamental role in the mobility of transposable elements and have been demonstrated to be of critical importance in Pelement insertion (Rubin and Spradling
1983). Since perfect inverted repeats are
characteristic of other active transposons,
such as the P-element of D. melanogaster
In Situ Hybridization
Procedures for preparation of Hessian fly
salivary polytene chromosomes were
modified from those developed for Drosophila polytene chromosomes (Pardue
1986) as described by Shukle and Stuart
(1994). The chromosomes were probed
with the whole mariner element (DesmarT)
labeled with [3H]TTP by oligonucleotide
random priming reaction (Feinberg and
Vogelstein 1983).
and Tel of Caenorhabditis elegans, it is in-
teresting to observe such a feature in the
Hessian fly mariner. In the case of mariner
elements, the requirements of transposition may not necessitate perfect inverted
repeats, since the mariner Mosl is functional, but the efficiency of transposition
might be affected by errors in recognition
sites in these regions. A comparison of
some of the reported terminal repeats
shows that there is considerable conservation of the sequence among a wide species range. Desmarl appears to share
Results and Discussion
Sequence Analysis of the Mariner
Element From the Hessian Fly
The recovered mariner element (designated DesmarT) is 1,288 bp in length, a size
A |_Aj G
T
G G [G | G T 1 G T| A A C/T TA T G A
A Q/T G O/A
G G T G T A C A A
A A A
G T G T A C A A C T
c
A A G T
T| A A A T
|c A A C T
consensus
T
C
A
G
G
T
G
T
R
C
A
A
T
A
A
C C
A A
A
A
A
A
A|G/O
A
A
C
C
C ] G
T
G
G| T
A|G
T G T
C C G
3/C C G
.C
c CC G
A
C| t
A
ATTA
C G Q/T|
G A A
G A A
G A A
T
T
TT
TT
TT
T T QIC
A
G A A A
C
C
G
G
A
A
T
T
Figure 2. A comparison of the Inverted repeats from the lacewing (Chrysoperia plorabunda; C. plor), a predatory mite (Metaseilus occidenlalis; M. occi), the frultfry (Drosophtla
mauritiana; Mosl), the Hessian fry (Mayetiota destructor, Desmarl), the ant (Myrmica ruginodis; M. rugi), the bumblebee (Bomous terrestris; B. terr), and the honeybee (Apis
mellifora; A. mell). When there are Inconsistencies In the Inverted repeat this Is Indicated with sequence of the 5' direction first followed by the complement of the 3'
sequence. NudeoUde Identities of these various species repeats with the Inverted repeats of Desmarl are enclosed by an outline. If there was Identity In one direction In the
Imperfect repeats the pair was Included within the outline.
Brief Communications
73
these elements was determined by in situ
hybridization to salivary gland polytene
T
G
chromosomes with the mariner element
G
G
Desmarl. While Desmarl is only one of
T
C
several mariner elements in the Hessian fly
genome, it does represent the predomiA
Desman
C
nant subfamily. Desmarl showed approxiA
G
M. occi
mately 50-80% homology to the other seFigure 3. A comparison of a conserved sequence Immediately following the Inverted repeats In the 5' direction
quences recovered by PCR products of
In the following species: Bombyx terrestris (B. terr), Myrmica ruginodis (M. rugl), Chrysoperia plorabunda (C. plor),
Drosophila mauntiana (Mosf). Mayetiola destructor (Desmarl), and Metaseilus ocadentalis (M. occi). The region of short length (300 bp), which represented
a highly conserved segment of the mariner
the conserved sequence Is enclosed In a box and Identical bases are Indicated In bold type.
transposase. It is possible that this probe
does not hybridize to all mariners present
in the genome. The resolution of the lamately
800
bases
flanking
each
side
of
the
no acid sequence for the transposase from
beling is limited in the Hessian fly when
the Hessian fly element showed 58% iden- mariner element, failed to align with any
sequences in the gene data bank, suggest- compared to in situ hybridization to Drotity and 72% similarity (Figure 4).
ing it might be an area of noncodlng DNA. sophila polytene chromosomes, because
A short region (49 bases) follows the
of the short size and reduced chromoThis
region does not appear to contain
stop codons and is characterized by a repalindromic sequences, as were demon- some morphology. Despite the limitations
peated CATT motif, followed by an A-rich
of the system, a general pattern of mariner
strated in hymenopteran species. Such a
region, and then the 3' inverted terminal
repeat. No pattern of sequence compara- conserved insertion site in the hymenop- localization with the Desmarl probe can
ble to the 5' internal flanking region was
teran order might be a mechanism to re- be seen. Autoradiographic label appears
to be predominantly localized over the
noted. This is in contrast to the situation
duce the possible mutagenic effect of
paracentromeric regions on the four chroseen in the P-element of Drosophila and
transposition by isolating such events in
mosomes, but there is also signal in other
which represented binding sites important
nonessential or repetitive DNA (Bigot et
areas. These patterns of localization were
for transposition (Kaufman et al. 1989).
al. 1994).
seen consistently in the spreads examined
The insertion of the mariner element in
and illustrated here by two or more repDistribution of Mariner Elements in the
the Hessian fly genome is flanked by TA
resentative samples of each chromosome
Hessian Fly Genome
both at the 5' and 3' ends. This may be a
(Figure 5). There are clear bands present
2 bp duplication at the site of insertion, Mariner elements have been estimated
on chromosome 1, both apical and distal
which is characteristic of other mariner el- through DNA blot analysis to be present
to the nucleolus on the long arm and a
ements (Hartl 1989). The region in which
in the Hessian fly genome in moderate
band distal to the centromere on the short
Desmarl has inserted into the Hessian fly
copy numbers similar to that in D. maun- arm. On chromosome 2 a distinct band
genome is AT rich. Sequence analysis of
tiana (20-30 copies) (Shukle and Russell
can be seen distal to a chromosome puff
the insertion site, consisting of approxi1995). The cytological distribution of
and bands are present near both ends.
B. terr
M. rugi
C. plor
Mos1
T
T G C C
G A G A
G A C A
C G A A C A
G G A
C G
C C
T A
A A
C A
T A
C
G A
T
C
T
T
T
T
A
A
A
A
A
A
G
G
G
G
G
G
A
A
A
A
A
A
T
T
T
T
T
G
G
G
G
G
G
G
G
G
G
T
T
G
C
C
C
C
C
C
c
C
C
T
C
A
T
T
A
C
G
C
T
De 1 MENFENWRKRRHLREVLLGHFFAKKTAAESHRLLVEVYGEHALAKTQCFE
cn
M F
+ + R VL+ F KKTAAESHR+LVE +GE+
C
Mo 1 MSSFV—PNKEQTRTVLIFCFHLKKTAAESHRMLVEAFGEQVPTVKTCER
51 WFQRFKSGDFDTEDKERPGQPKKFEDEELEALLDEDCCQTQEELAKSLGV
WFQRFKSGDFD +DKE+
PK++ED EL+ALLDED QTQ++LA L V
4 9 WFQRFKSGDFDVDDKEHGKPPKRYEDAELQALLDEDDAQTQKQLAEQLEV
101 TQQAISKRLKAAGYIQKQGNWVPHELKPRDVERRFCMSEMLLQRHKKKSF
+QQA+S RL+ G IQK G WVPHEL R +ERR
E+LL R+K+KSF
99 SQQAVSNRLREMGKIQKVGRWVPHELNERQMERRKNTCEILLSRYKRKSF
151 LSRIITGDEKWIHYDNSKRKKSYVKRGGRAKSTPKSNLHGAKVMLCIWWD
L RI+TGDEKWI + N KRKKSYV G A ST++ N G K MLC+WWD
14 9 LHRIVTGDEKWIFFVNPKRKKSYVDPGQPATSTARPNRFGKKTMLCVWWD
201 QRGVLYYELLEPGQTITGDLYRTQLIRLKQALAEKRPEYAKRHGAVIFHH
Q GV+YYELL+PG+T+
Y+ QLI L +AL KRPEY KR+ VIF H
199 QSGVIYYELLKPGETVNTARYQQQLINLNRALQRKRPEYQKRQHRVIFLH
251 DNARPHVALPVKNYLENSGWEVLPHPPYSPDLAPSDYHLFRSMQNDLAGK
DNA H A +V++ LE
WEVLPH++YSPDLAPSDYHLF SM + LA +
24 9 DNAPSHTARAVRDTLETLNWEVLPHAAYSPDLAPSDYHLFASMGHALAEQ
301 RFTSEQGIRKWLDSFLAAKPAKFFEKGIHELSERWEKVIASDGQYFE
RF S + ++KWLD ++AAK +F+ +GIH+L ERWEK +ASDG+YFE
299 RFDSYESVKKWLDEWFAAKDDEFYWRGIHKLPERWEKCVASDGKYFE
347
345
Figure 4. A comparison of the putative translation of the open reading frames of the mariner transposases of
the Hessian fly, Desmarl (De), with that of the fruit fly, Drosophila mauritiana, MosI (Mo), showing the consensus
amlno acids (Cn). The + sign Indicates a conservative substitution. The MosI sequence Is modified from Genbank
accession no. M14653 (Medhora et al. 1991). Desmar Genbank accession no. Is U24436.
7 4 The Journal erf Heredity 1997:88(1)
Chromosomes 3 and 4 show label predominantly near the centromeres and there is
a suggestion of a second band on chromosome 3. Paracentromeric regions are
heterochromatic and contain repetitive
DNA; features characteristic of noncoding
regions of the genome. The localization of
mariner elements in heterochromatin is
similar to the localization seen in several
other transposable elements, but is in distinct contrast to the observation reported
for /"-elements, which are predominantly
in euchromatin (Engels 1989). While several single bands indicate mariner sites
outside heterochromatic regions in the
Hessian fly genome, and while mariner was
first discovered in D. mauritiana because
of an insertion into an eye-color gene, a
preponderance of paracentromeric insertion sites could indicate that mariner is
usually sequestered into noncritical
regions of the chromosome. There would
be some selective pressure for such a
mechanism since too-frequent transposition events into coding regions would be
dysgenic. Before the mariner element can
10M
Figure 5. In situ hybridization of Desmarl to the lour salivary gland polytene chromosomes of Mayeliola destructor. Each chromosome is Indicated by number and each Is
represented by at least two examples from different Individuals. The nucleolus of chromosome 1, located on the long arm, Is Indicated by N, p indicates a puff on the short
arm of chromosome 2, c Indicates paracentromeric regions which show hybridization to Desmarl, black arrows indicate locations of consistent hybridization to probe other
than at paracentromeric regions, and open arrows Indicate probable areas of hybridization. Total magnification for all chromosomes is l,400x.
be a useful tool for genetic manipulation,
such a mechanism must be understood.
Furthermore, studies of mariner elements
in Drosophila have indicated that the position of the element in the genome and
the immediate adjacent flanking sequences are of profound importance in determining its transposing activity (Medhora
et al. 1991). More sites must be examined
by sequence analysis before any generalizations can be made concerning preferential insertion of mariner elements in the
Hessian fly.
The presence of perfect inverted repeats and the identity shared by the transposase of the Hessian fly element with
that of the autonomous element Mosl sug-
gest that Hessian fly mariner element
might also be autonomous. Work is currently being undertaken to assess the biological activity of this element.
site and distribution. Proc Natl Acad Scl USA 91:34083421.
From the Department of Entomology, Purdue University, West Lafayette, IN 47907 (Russell and Shukle), and
the Agricultural Research Service, USDA, Purdue University (Shukle). Purdue University AES Journal paper
14649. Supported through USDA CRIS no. 3602-2200008-OOD. Mention of a commercial or proprietary product does not constitute an endorsement by the USDA.
Address correspondence to V. W. Russell at the address
above.
Engels WR, 1989. P elements In Drosophila melanogaster. In: Mobile DNA (Berg DE and Howe MM, eds).
Washington: American Society for Microbiology, 437484.
Ebert PR, Hlleman JP, and Nguyen HT, 1995. Primary
sequence, copy number, and distribution of mariner
transposons In the honey bee. Insect Mol Blol 4:69-78.
Felnberg A and Vogelsteln B, 1983 A technique for radlolabellng DNA restriction endonuclease fragment to
high specific activity. Analyt Biochem 132:6-13.
The Journal of Heredity 199758(1)
Hart! DL, 1989. Transposable element mariner In Drosophila species. In: Mobile DNA (Berg DE and Howe
MM, eds). Washington: American Society for Microbiology; 531-537.
References
Bigot Y, Hamelln M, Capy P, and Perlquet G, 1994. Marmcr-Uke elements In hymenopteran species: Insertion
Jacobson JW, Medhora MM, and Hartl DL, 1986. Molecular structure of a somaticalry unstable transposable
element In Drosophila Proc Natl Acad Sd USA 83:86848688.
Brief Communications 7 5
Jeyaprakash A and Hoy MA, 1995. Complete sequence
of a manner transposable element from the predatory
mite Metasewtus occidentalis Isolated by an inverse
PCR approach. Insect Mol Blol 4:31-39.
Kaufman PD, Doll RF, and Rio DC, 1989. Drosophila P
element transposase recognizes Internal P element
DNA sequences Cell 59:359-371.
Us JT, Simon JA, and Sutton CA, 1983. New heat shock
puffs and fJ-galactosldase activity resulting from transformation of Drosophila with an hspT04ac2. hybrid
gene. Cell 35:403-^10.
Maruyama K, Schoor KD, and Hartl DL, 1991. Identification of nucleotide substitutions necessary for transactivation of mariner transposable elements In Drosophila: analysis of naturally occurring elements. Genetics 128:777-784.
Medhora M, Maruyama K, and Hartl DL, 1991. Molecular and functional analysis of the mariner mutator element Most In Drosophila. Genetics 128:311-318.
Pardue ML, 1986. In situ hybridization to DNA. In: Drosophila, a practical approach (Roberts DB, ed.). Washington: IRL Press; 111-137.
Robertson HM, 1993. The mariner transposable element is widespread in Insects. Nature 362:241-245.
Robertson HM and Lampe DJ, 1995. The distribution of
transposable elements In arthropods. Ann Rev Entomol
40:333-357.
Robertson HM and MacLeod EG, 1993. Five major subfamilies of mariner transposable elements In Insects,
Including the Mediterranean fruit fly, and related arthropods. Insect Mol Blol 2:125-139.
Rubin GM and Spradling AC, 1983. Vectors for P-element-medlated gene transfer In Drosophila. Nucleic Acids Res 11.6341-6351.
Sanger F, Nlcklen S, and Coulson AR, 1977. DNA sequencing with chain terminating Inhibitors. Proc Natl
Acad Sci USA 745463-5467.
available, and the reported pressures
have not proven effective. Here, microsatellite markers were employed in a redetermination of the pressures required to generate zebrafish half-tetrads with currently
available equipment. Two criteria were
used to choose conditions for half-tetrad
gynogenesis: expected ploidy as confirmed with microsatellite markers and
maximal egg viability. Pressures between
6140 and 6840 psi generate half-tetrad
embryos, with optimal viabilities attained
at 6140 and 6240 psi. As predicted, early
pressure treatment of eggs fertilized with
nonirradiated sperm created triploid embryos.
years (Streisinger et al. 1981). These and
other experimental features of the zebrafish have lately made possible the production of hundreds of developmental mutants, which increases the urgency of genetic mapping (Mullins et al. 1994; SolnicaKrezel et al. 1994). Genetic mapping in
turn is assisted in the zebrafish by the
ability to produce half-tetrad embryos
(Streisinger et al. 1981).
The zebrafish (Danio rend) is an attractive
experimental system for a variety of reasons. The transparency of the embryos,
easy collection of large numbers of embryos, and development ex vivo allow the observation of development in great detail
(Concordet and Ingham 1994; Kahn 1994;
Strahle and Ingham 1992). The fecundity
of the zebrafish makes screening for mutants practical. Sexual maturity is attained
in 3-4 months, and the fish live about 2
Half-tetrad embryos are produced by activation of oocyte cell division with UVirradiated sperm, followed by disruption
of microtubule-mediated meiosis II disjunction (meiosis II normally occurs postfertilization) using hydraulic pressure
(early pressure, or EP) (Streisinger et al.
1981, 1986) (Figure 1). Pigment phenotypes of half-tetrad embryos were used by
Streisinger et al. (1981) and Johnson et al.
(1995) to calculate marker-centromere distances for recessive pigment markers
(Johnson et al. 1995; Streisinger et al.
1986). While attempting to generate halftetrad embryos using EP, we found that
the original equipment was no longer
available and that the reported pressure
of 8000 psi (Streisinger et al. 1981; Wester-
No
Crossover
Crossover
Shukle RH and Stuart JJ, 1993. A novel morphological
mutation In the Hessian fly, Magetiola destructor. J Hered 84:229-232.
Shukle RH and Stuart JJ, 1995. Physical mapping of
DNA sequences In the Hessian fly, Mayeliola destructor.
J Hered 86:1-5.
Shulde RH and Russell VW, 1995. Afar/ner-like sequences from the Hessian Dy, Mayetiola destructor. 1 Hered
86:364-368.
Received November 28, 1995
Accepted May 20, 1996
Corresponding Editor: Ross Maclntyre
Meiosis I
•M
Homozygous
d
New Conditions for
Generation of Gynogenetic
Half-Tetrad Embryos in the
Zebrafish (Danio rerio)
\
\
\
E. E. Gestl, E. J. Kauffman, J. L.
Moore, and K. C. Cheng
The generation of gynogenetic half-tetrads
is an important tool for genetic mapping
and mutant screens in zebrafish (Danio rerio). Half-tetrad gynogenesis can be accomplished using hydraulic pressure to
disrupt microtubule-mediated segregation
during meiosis II, which normally occurs
after fertilization. However, the equipment
used in the original studies is no longer
7 6 Trie Journal of HereoSty 1997:88(1)
' H&tHMrad imbiyot
Figure 1. Early pressure parthenogenesis yields half-tetrad embryos. In each Intermediate, the chromosome
configuration at a hypothetical locus A Is drawn. Alleles A and a represent microsatellite alleles which contain
more (A), or fewer (a) repeat units. PCR amplification of this locus followed by electrophoretlc fractlonatlon of
the products and autoradlography yields the schematlclzed gel bands shown below each corresponding intermediate. No crossovers or an even number of crossovers between a marker and Its centromere yields homozygous
half-tetrad genotypes (* under "No crossovers"); an odd number of crossovers between a marker and Its centromere yields heterozygous half-tetrad genotypes. Early pressure parthenogenesis Inhibits the segregation of sister
chromatlds that would normally occur during meiosis II, as Indicated on the bottom left. The genotypes of the
secondary oocytes (*) are Identical to those of the hali-tetrads generated by EP parthenogenesis. Reproduced with
permission from Academic Press.