Cell Recognition and Synapse Formation

Nirenberg, Marshall W.

Abstract

DNA fragments -0.57, -2.2, -2.9, -5.3, and -8.4 kb in length from the regulatory region of the vnd/NK-2 gene were cloned in the 5'- flanking region of a beta-galactosidase(b-gal) reporter gene in the P-element pCaSpeR-AUG-b-gal, and the effects of the DNA inserts on the pattern and time of expression of b-gal were determined in transgenic Drosophila embryos. The results showed that the -8.4 kb DNA fragment contains the nucleotide sequences required to generate the normal pattern of vnd/NK-2 gene expression, and that -5.3 to -8.4 kb DNA is required for vnd/NK-2 gene expression in the most dorsoanterior part of the cephalic region. No partial pattern of vnd/NK-2 gene expression was detected with smaller fragments of DNA tested. vnd/NK-2 mutant embryos containing the homozygous P-element p[-8.4 to + 0.34 b-gal] expressed little or no beta-gal in contrast to embryos with a wild-type vnd/NK-2 gene. Therefore, vnd/NK-2 protein is required, directly or indirectly, for maintenance of vnd/NK-2 gene expression. In a collaborative study with James Ferretti and his associates, the three dimensional structure of the vnd/NK-2 homeodomain was determined. The effects of mutations in positions 52, 54, and 56 of the vnd/NK-2 homeodomain on the secondary structure of the homeodomain and the ability of the homeodomain protein to bind to DNA were determined. Three single sites H52R, Y54M, and T56W mutations, 2 double site H52R/T56W and Y54M/T56W mutations, and one triple site H52R/Y54M/T56W mutations were investigated. Replacement of Y54 with M resulted in a ten- fold decrease in the affinity of the homeodomain for DNA containing the vnd/NK-2 consensus sequence, CAAGTG. T56 plays an important role in determining the length of the DNA recognition helix. Replacement of H52 with R does not affect the length of the helix but does increase the thermal stability. Replacement of A35 by T is a lethal mutation in Drosophila. The mutant homeodomain was shown to be unable to adopt a folded conformation. The affinity of the A35T mutant vnd/NK-2 homeodomain for the vnd/NK-2 target DNA sequence is 50-fold lower than that of the wild-type homeodomain.

Funding Agency

• Agency: National Institute of Health (NIH)
• Institute: National Heart, Lung, and Blood Institute (NHLBI)
• Type: Intramural Research (Z01)
• Project #: 1Z01HL000009-23
• Application #: 6109134
• Study Section: Special Emphasis Panel (LBG)
• Support Year: 23
• Fiscal Year: 1998

Institution

• Name: National Heart, Lung, and Blood Institute
• Country: United States

Publications

Xia, Menghang; Huang, Ruili; Guo, Vicky et al. (2009) Identification of compounds that potentiate CREB signaling as possible enhancers of long-term memory. Proc Natl Acad Sci U S A 106:2412-7

Sharma, Shail K; Nirenberg, Marshall (2007) Silencing of genes in cultured Drosophila neurons by RNA interference. Proc Natl Acad Sci U S A 104:12925-30

Koizumi, Keita; Higashida, Haruhiro; Yoo, Siuk et al. (2007) RNA interference screen to identify genes required for Drosophila embryonic nervous system development. Proc Natl Acad Sci U S A 104:5626-31

Chen, Xiaobing; Vinade, Lucia; Leapman, Richard D et al. (2005) Mass of the postsynaptic density and enumeration of three key molecules. Proc Natl Acad Sci U S A 102:11551-6

Ivanov, Andrej I; Rovescalli, Alessandra C; Pozzi, Paola et al. (2004) Genes required for Drosophila nervous system development identified by RNA interference. Proc Natl Acad Sci U S A 101:16216-21

Lum, Lawrence; Yao, Shenqin; Mozer, Brian et al. (2003) Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells. Science 299:2039-45

Nirenberg, Marshall (2003) The genetic revolution: the importance of flies and worms. Am J Psychiatry 160:615

Koizumi, Keita; Lintas, Carla; Nirenberg, Marshall et al. (2003) Mutations that affect the ability of the vnd/NK-2 homeoprotein to regulate gene expression: transgenic alterations and tertiary structure. Proc Natl Acad Sci U S A 100:3119-24

Hwang, Kae-Jung; Xiang, Bosong; Gruschus, James M et al. (2003) Distortion of the three-dimensional structure of the vnd/NK-2 homeodomain bound to DNA induced by an embryonically lethal A35T point mutation. Biochemistry 42:12522-31

Wang, Lan-Hsiang; Chmelik, Rebecca; Nirenberg, Marshall (2002) Sequence-specific DNA binding by the vnd/NK-2 homeodomain of Drosophila. Proc Natl Acad Sci U S A 99:12721-6