2092 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / December 1987 Vol. 28

A DNA Deletion Associated With Multiple ImpairedTranscripts in the Visual Mutant TRP

Ming Lu and Fulron Wong

The transient receptor potential (trp) mutant in Drosophilais known to manifest retinal degeneration and involves de-fects in the intermediate steps of visual transduction. Thechromosome walking technique has been conducted at thecytogenetic location of trp. Overlapping phage clones thatcover a DNA stretch of 73 kb were obtained. Southernblotting studies detected a 2.3 kb DNA deletion within thisregion in the mutant. The deleted DNA fragment containsexons of three transcripts with lengths of 3.5, 1.5 and 0.8kb. The transcription of the 3.5 and 1.5 kb RNAs is com-pletely abolished in the mutant, whereas that of 0.8 kbRNA appears to be modified. The impairment in productionof three RNAs, due to the DNA deletion, offers a possiblemolecular basis of the multiple defects displayed by the trp~phenotype. Invest Ophthalmol Vis Sci 28:2092-2095,1987

The transient receptor potential {trp) mutant inDrosophila melanogaster was first isolated on thebasis of its behavioral phenotype.1 The effect of thetrp mutation occurs within the as of yet undefinedmechanisms which link light-induced changes inrhodopsin to subsequent membrane conductancechanges. The term "intermediate steps" of transduc-tion has been used to describe these undefined mech-anisms. In this regard, the trp mutation has specialsignificance because it is one of the few mutants iso-lated so far that is thought to affect these intermediatesteps of visual transduction.2 In addition to permit-ting the elucidation of a model for transduction, thetrp mutation is also important for the study of heredi-tary retinal abnormalities. The physiological defect,due to the trp mutation, leads to retinal degenerationin the adult. These symptoms parallel in several re-spects some of those observed in human pathologicalconditions. A common complaint of patients withretinitis pigmentosa is "night-blindness." A muchmore severe complaint is that light causes "white-out" and loss of visual function for extended periods.These observations have suggested that aspects of ret-inal function related to visual transduction are in-volved.3

In a previous communication,4 we reported theabsence of two RNA transcripts with lengths of 1.5and 3.5 kb in the trp mutant. DNA structure changesassociated with the trp mutation have previously notbeen determined. This article describes a 2.3 kb DNAdeletion in the trp mutant within a 73 kb chromo-somal walking region at the cytogenetic location oftrp. The deleted DNA fragment appears to retaincoding sequences for three distinct mRNAs. Our

findings suggest that the multiple effects of the trpmutant may reflect the impairment of multiple tran-scripts from the trp locus.

Materials and Methods. A Drosophila genomicDNA library5 was probed with DNA fragments la-beled with 32P-dCTP (3,000 Ci/mmole, Amersham,Arlington Heights, IL). The procedure known aschromosome walking6 was used to isolate genomicDNA adjacent to specific DNA fragments or probeswhich were previously isolated. Plaques (approxi-mately 30,000 per 150 mm Petri plate) were trans-ferred to nitrocellulose. The filters were baked for 2 hrat 80°C under vacuum. They were then prehybrid-ized for 1-2 hr at 65 °C with 0.6 M NaCl, 8 mMEDTA, 0.12 M Tris-Cl (pH 8), and 2X Denhardt'ssolution, and finally hybridized with the 32P-labeledprobes in prehybridization buffer plus 0.1 mg/mlsheared salmon sperm DNA at 65°C for 16-24 hr.The filters were washed once for 10 min duration at20°C with 0.15 M NaCl, 0.17 M Na citrate (pH 7),and 0.1% SDS, then three times for 30 min durationsat 65°C in 0.45 M NaCl, 0.09 M Tris-Cl, 6 mMEDTA (pH 8) and 0.2% SDS and finally for 30 min at65°C with 0.15 M NaCl, 0.03 M Tris-Cl, 2 mMEDTA (pH 8) and 0.2% SDS. Following air dryingthe filters were exposed to Kodak XAR-5 film withan intensifying screen for 3-24 hr at -70°C.

Genomic DNA for blotting was prepared fromadult flies according to the procedure described inKuner et al.7 Restriction endonuclease digests of ge-nomic DNA and cloned DNA were electrophoresed,along with size markers (restriction digests of lambdaDNA), for 16 hr at 1.1 V/cm in 0.7% agarose gels inTAE buffer. Each lane contained 1-10 Mg of DNA.The DNA was transferred to nitrocellulose. The la-beled DNA probes were hybridized to the DNA blotsas described above for plaque filter hybridization.

DNA fragments for subcloning were separated byelectrophoresis in agarose gels, followed by electro-elution in TAE buffer. The eluate was passed throughan Elutip-d column (Schleicher and Schuell, Keene,NH) and the DNA was precipitated with ethanol. TheDNA fragments were then cloned into the appro-priate restriction sites in pBR322.

Drosophila RNA was prepared using the proceduredescribed in Wong et al.4 Poly(A)+RNA was purifiedwith an oligo(dT) column.8 RNA was electropho-resed for 16 hr at 1.1 V/cm in 1.4% agarose gels con-taining 2.2 M formaldehyde. Mouse liver and E. coli

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No. 12 Reporrs 2093

ribosomal RNA were used as size markers. The sepa-rated Drosophila RNA was transferred to nitrocellu-lose, blotted, and hybridized with labeled probes. Inorder to calibrate the amount of RNA in each lane,signals resulting from hybridization using a DNAprobe which contains an actin sequence were com-pared to those obtained with other DNA probes.

Results. A genomic clone 559 was initially mappedto 99C 5-6 on the right arm of the third chromosomeof Drosophila.9 Our unpublished in situ hybridiza-tion results show that it resides in the cytogeneticlocation of trp, which is denned by a pair of duplica-tion-deletion strains.10 Chromosomal walk stepsfrom both sides of clone 559 have been characterized(see Fig. 1). By using clone 559 as probe, eight clonesthat partially overlap with clone 559, including clones7A1, 1F2 and 1A1, were initially obtained from thegenomic library. Restriction fragments from clones7A1 and 1F2, both of which were most distant fromclone 559, were used as probes in subsequent repeti-tions of the procedure to obtain, respectively, clonesincluding 4A and 14. Appropriate fragments ob-tained from these "steps," in turn, were used asprobes and they yielded respectively clones 52 andT2. The extent of overlap of neighboring clones wasdetermined by cross-hybridizations.

For comparison, total genomic DNA extractedfrom homozygous mutant and from normal flies wasdigested with a set of restriction enzymes, electropho-resed, blotted, and hybridized, using as probes indi-vidual clones obtained from chromosome walking. Inmost cases identical hybridization patterns were ob-served between the genomic DNA of mutant and thenormal flies. However, when clone 7A1 was used tohybridize £coRI digested genomic DNA blots, differ-ent hybridization patterns were detected. The geno-mic DNA blot from normal flies yields six DNAbands with sizes of 1.8, 2.7, 3.5, 5.0, 5.1 and 11.6 kb(Fig. 2a, lane 1), whereas the blot from the mutantgives five identical bands as those of the normal flyand an aberrant sixth band of 9.3 kb (Fig. 2a, lane 2).This DNA structure change identified by probe 7A Isuggests a deletion in the trp mutant, as no additionalbands were detected. This DNA structure change wasalso observed when four other enzymes were used todigest the genomic DNA. One of them in particular,BamHl, revealed a deletion of a single 2.3 kb band inthe trp genomic DNA (Fig. 2b).

The 2.3 kb deleted DNA was isolated from clone4A of the normal flies and subcloned asymmetricallyas two BamHl-Sall fragments (Fig. 1). The resultingsubclones, designated BS32 (1.0 kb insert) and BS60(1.3 kb insert), were used as probes to hybridize toBamHl digested genomic DNA from the mutant andfrom the normal flies. The resulting patterns con-

1 19 2

BSSO

\ ,

i

4A

BS33

1

1 1

| |

7A1

599

I I |

(

1A1

i l l i14

1 l 11F2

1 |

1 1

10 kb

genomic

O N *

T2

Fig. 1. A schematic diagram of the relationship of a set of eightoverlapping genomic clones generated by EcoRl restriction (verti-cal lines) that spans 73 kb of DNA in the trp region. B, BamHl site;S, Sail site. The 2,3 kb deletion identified by probe 4A was sub-cloned as two BamHl-Sall fragments: BS60 (1.3 kb) and BS32 (1.0kb).

firmed that the 2.3 kb band was missing from the trpmutant (Fig. 3). The probes also identified otherbands which were present in normal as well as trpflies. These observations suggest that there is homol-ogy between the 2.3 kb DNA sequence and otherDNA fragments in and outside the trp locus.

The two probes BS32 and BS60 were subsequently

p 2.3kb

Fig. 2. DNA structure change identified by different probes ongenomic DNA blots, (a) One of the six EcoRl restriction fragmentsidentified by probe 7AI shows DNA structure change, 11.6 kb innormal flies (Oregon-R strain, lane 1) versus 9.3 kb in the mutant(lane 2). (b) Probe 4A identified seven BamHl restriction fragmentsof 1.6, 2.1, 2.3, 3.0, 4.1, 9.0 and 10 kb in normal flies (lane 1). Oneof the seven DNA bands (2.3 kb) is missing in the mutant (lane 2).

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2094 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / December 1987 Vol. 28

3 4 right arm of the third chromosome. Clone 559 whichoverlaps with the cytogenetic location of trp was usedas probe for chromosome walking, in such a way thatpartially overlapping DNA clones that cover a DNAstretch of 73 kb were obtained (Fig. 1). The Southernblotting study of the 73 kb chromosomal walkingregion reveals a DNA deletion in the mutant (Fig. 2).Furthermore, subclones BS32 and BS60, derivedfrom the deleted DNA fragment, hybridize to a DNAband in normal flies that is absent in the mutantDNA, suggesting that the 2.3 kb fragment is a simpledeletion and that other complications on the DNA

« - 2 . 3 k b

Fig. 3. The identified 2.3 kb deleted DNA band was subclonedfrom clone 4A as two fragments. These fragments were individuallyused as probes to hybridize BamHl digested genomic DNA blots:BS60 (lanes 1 and 2) and BS32 (Lanes 3 and 4). BS60 identified the2.3 kb band in the normal fly (lane 1), but not in the mutant (lane2). The same patterns were observed by using BS32 as probe tohybridize the normal fly (lane 3) and mutant DNA (lane 4). Theprobes identified other bands suggesting homology between the 2.3kb DNA and the additional DNA such as the 2.1 kb band.

used to hybridize to RNA extracted from normal flies(Fig. 4). Probe BS60 identified one RNA band of 0.8kb (lane 1) and probe BS32 identified three RNAbands of 3.5, 1.5 and 0.8 kb (lane 2).

Discussion. The trp mutation in Drosophila me-lanogaster is recessive. Although the final productsfor the trp gene(s) are not known, the cytogeneticlocation of trp has been mapped to 99C 5-6 on the

3.5

* 1.5

0.8

Fig. 4. RNA bands from the normal fly identified by probes BS60(lane I) and BS32 (lane 2), The sizes of the mRNAs are indicated inkbs.

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No. 12 Reports 2095

structure are not involved (Fig. 3). This observedDNA structure change probably defines the primarydefect associated with the mutation. The deletedfragment identifies three RNAs on northern blots ofRNA of normal flies (Fig. 4). Some coding sequencesin these transcripts are probably missing in the mu-tant (Fig. 4). It is concluded that the protein productsthey encode are also affected. Specifically, two pro-teins are probably missing because two mRNAs (3.5and 1.5 kb respectively) are missing4 and the proteinencoded by the 0.8 kb transcript may be altered be-cause some of its exons have been deleted in the mu-tant. The abnormality of more than one transcript,due to the deletion, may result in the numerous de-fects associated with the trp mutant.

The 3.5, 1.5 and 0.8 kb RNAs are independentstable transcripts, since we have obtained cDNAclones complementary to each of these RNAs. Wehave also hybridization-selected these mRNAs fromnormal fly total RNA and translated them in vitrointo three distinct proteins of 107,66 and 33 kD (datanot shown). Therefore, these transcripts are not pre-cursors or degraded products of each other. The dele-tion in the mutant did not significantly alter the sizeor the expression of the 0.8 kb transcript as the corre-sponding band appears in similar amounts and at thesame position as in that of the normal fly.4 A fulllength cDNA clone complementary to the 0.8 kbtranscript was used to hybridize EcoKl and BamHldigested blots of genomic clones obtained from chro-mosome walking (unpublished results). Exons of 0.8kb RNA were determined to be localized within agenomic DNA stretch of 9 kb. The cDNA clone hy-bridizes to the deleted fragment along with severalother DNA bands that flank the 2.3 kb band. Sincethe cDNA clone identifies the deleted fragment andthe 2.1 kb adjacent DNA band, it suggests that bothDNA fragments contain exon(s) for the 0.8 kb tran-script. Furthermore, subclone BS60 identifies the 0.8kb transcript as the sole RNA band in the northernblot (Fig. 4), strongly indicating that the deleted DNAfragment contains exon(s) for the 0.8 kb transcript.

Earlier attempts were made to study the muta-tional defects of trp by looking for alterations in mo-lecular weight and isoelectric point of proteins in themutant. The major difficulty associated with this ap-proach is that a simple mutation may cause changesin many proteins, often making it impossible to dis-tinguish the primary effect of the mutation from thesecondary ones. Identification of any alterations inDNA of the mutant is simpler to interpret. As alter-ations in DNA can be identified, the mutant pheno-type can be specifically ascribed to modifications inthe ultimate gene product, the protein. Althoughthere could still be multiple changes resulting from

the mutation, a direct causal relationship for the pri-mary effect can be established. For example, the se-quence of these mRNA transcripts can be determinedso that the corresponding amino acid sequences canbe identified. The amino acid sequences derived canbe compared with all known protein sequences. If it isrevealed that the proteins have homologous peptideswith some familiar proteins, they may also have anal-ogous functions. The sequence information wouldalso allow the production of antibodies targeted bydesign at unique sites of the proteins for studying thelocalization, function and developmental expressionof the proteins.Key words: visual transduction, retinal abnormality, DNAdeletion, mutational defect, multiple transcripts

Acknowledgments. The authors thank Peggy Chang fortechnical assistance. The generosity of Dr. Ross Maclntyreof Cornell University in providing the duplication-deletionstocks and Dr. Michael Goldberg of Cornell University inproviding the Drosophila genomic library is gratefully ac-knowledged.

From the Marine Biomedical Institute, University of Texas Med-ical Branch, Galveston, Texas. Submitted for publication: January26, 1987. Reprint requests: Ming Lu, PhD, Department of Ge-netics, The Hospital for Sick Children, 555 University Avenue,Toronto, Ontario, Canada M5G 1X8.

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8. Maniatis T, Fritsch EF, and Sambrook J: Molecular Cloning, aLaboratory Manual. Cold Spring Harbor, New York, ColdSpring Harbor Press, 1982.

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