Isolation of genes encoding photoreceptor-specific proteins by immunoscreening with antibodies directed against purified blowfly rhabdoms

Autoren

Armin Huber
Philipp Sander
Uwe Wolfrum
Christine Groell
Gabriele Gerdon
Reinhard Paulsen

DOI

10.1016/1011-1344(96)07310-1

ISSN

1011-1344

Ausgabe der Veröffentlichung

1-2

Zeitschrift

Journal of Photochemistry and Photobiology B: Biology

Sprache

en

Paginierung

69 - 76

Datum der Veröffentlichung

1996

Status

Published

Herausgeber

Elsevier BV

Herausgeber URL

http://dx.doi.org/10.1016/1011-1344(96)07310-1

Datum der Datenerfassung

2021

Titel

Isolation of genes encoding photoreceptor-specific proteins by immunoscreening with antibodies directed against purified blowfly rhabdoms

Ausgabe der Zeitschrift

35

Data source: Crossref

Abstract

The proteins which perform and regulate key steps in phototransduction are assumed to be localized in the rhabdomeric membrane of invertebrate photoreceptor cells. We have employed antibodies raised against rhabdoms purified from blowfly eyes in order to isolate copy deoxyribonucleic acid (cDNA) clones encoding proteins that are required in the phototransduction machinery. By immunoscreening a Calliphora retinal cDNA library, we obtained clones of genes coding for five different proteins. As revealed by partial cDNA sequence analysis, three of these genes represent the Calliphora homologs of Drosophila trp, inaC and InaD, while the other two displayed no homology to known genes. Northern blot analysis confirmed that trp, inaC and InaD transcripts were present in RNA isolated from the retina, but not in RNA isolated from brain or thorax. Specific antibodies directed against trp, inaC and InaD protein were raised using recombinantly expressed proteins or synthetic peptides. Western blot analyses revealed that trp, inaC and InaD protein are specifically associated with the rhabdomeral photoreceptor membrane. Extraction of membranes with buffers of different ionic strengths suggested that the trp gene product is an integral membrane protein, whilst the inaC and InaD gene products are peripherally bound membrane proteins. This demonstrates that the immunoscreening approach used here can be successfully applied to isolate genes that code for either integral or peripheral photoreceptor membrane proteins.

Addresses

Institute of Zoology I, University of Karlsruhe, Germany.

Autoren

A Huber
P Sander
U Wolfrum
C Groell
G Gerdon
R Paulsen

DOI

10.1016/1011-1344(96)07310-1

eISSN

1873-2682

Externe Identifier

PubMed Identifier: 8823935

Open access

false

ISSN

1011-1344

Ausgabe der Veröffentlichung

1-2

Zeitschrift

Journal of photochemistry and photobiology. B, Biology

Schlüsselwörter

Animals
Diptera
Insect Hormones
Calcium Channels
Eye Proteins
Insect Proteins
Drosophila Proteins
DNA, Complementary
RNA, Messenger
Fluorescent Antibody Technique, Indirect
Blotting, Western
Sequence Analysis, DNA
Antibody Specificity
Gene Expression
Amino Acid Sequence
Sequence Homology, Amino Acid
Molecular Sequence Data
Male
Transient Receptor Potential Channels
Photoreceptor Cells, Invertebrate

Sprache

eng

Medium

Print

Paginierung

69 - 76

Datum der Veröffentlichung

1996

Status

Published

Datum der Datenerfassung

1996

Titel

Isolation of genes encoding photoreceptor-specific proteins by immunoscreening with antibodies directed against purified blowfly rhabdoms.

Sub types

Research Support, Non-U.S. Gov't
Journal Article

Ausgabe der Zeitschrift

35

Data source: Europe PubMed Central

Abstract

The proteins which perform and regulate key steps in phototransduction are assumed to be localized in the rhabdomeric membrane of invertebrate photoreceptor cells. We have employed antibodies raised against rhabdoms purified from blowfly eyes in order to isolate copy deoxyribonucleic acid (cDNA) clones encoding proteins that are required in the phototransduction machinery. By immunoscreening a Calliphora retinal cDNA library, we obtained clones of genes coding for five different proteins. As revealed by partial cDNA sequence analysis, three of these genes represent the Calliphora homologs of Drosophila trp, inaC and InaD, while the other two displayed no homology to known genes. Northern blot analysis confirmed that trp, inaC and InaD transcripts were present in RNA isolated from the retina, but not in RNA isolated from brain or thorax. Specific antibodies directed against trp, inaC and InaD protein were raised using recombinantly expressed proteins or synthetic peptides. Western blot analyses revealed that trp, inaC and InaD protein are specifically associated with the rhabdomeral photoreceptor membrane. Extraction of membranes with buffers of different ionic strengths suggested that the trp gene product is an integral membrane protein, whilst the inaC and InaD gene products are peripherally bound membrane proteins. This demonstrates that the immunoscreening approach used here can be successfully applied to isolate genes that code for either integral or peripheral photoreceptor membrane proteins.

Autoren

A Huber
P Sander
U Wolfrum
C Groell
G Gerdon
R Paulsen

Autoren-URL

https://www.ncbi.nlm.nih.gov/pubmed/8823935

DOI

10.1016/1011-1344(96)07310-1

ISSN

1011-1344

Ausgabe der Veröffentlichung

1-2

Zeitschrift

J Photochem Photobiol B

Schlüsselwörter

Amino Acid Sequence
Animals
Antibody Specificity
Blotting, Western
Calcium Channels
DNA, Complementary
Diptera
Drosophila Proteins
Eye Proteins
Fluorescent Antibody Technique, Indirect
Gene Expression
Insect Hormones
Insect Proteins
Male
Molecular Sequence Data
Photoreceptor Cells, Invertebrate
RNA, Messenger
Sequence Analysis, DNA
Sequence Homology, Amino Acid
Transient Receptor Potential Channels

Sprache

eng

Country

Switzerland

Paginierung

69 - 76

PII

1011-1344(96)07310-1

Datum der Veröffentlichung

1996

Status

Published

Datum, an dem der Datensatz öffentlich gemacht wurde

1996

Titel

Isolation of genes encoding photoreceptor-specific proteins by immunoscreening with antibodies directed against purified blowfly rhabdoms.

Sub types

Journal Article
Research Support, Non-U.S. Gov't

Ausgabe der Zeitschrift

35

Data source: PubMed

Isolation of genes encoding photoreceptor-specific proteins by immunoscreening with antibodies directed against purified blowfly rhabdoms

Tools