This abstract for a paper to be delivered at Cold Spring Harbor explains the role of NK-2 homeobox genes in early development.
NK-2 is expressed initially by nuclei that are precursors of neuroectodermal cells, which in turn give rise to other major
parts of the nervous system.
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1997-07-28 (July 28, 1997)
Tsao, D. H.H.
Nirenberg, Marshall W.
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L.-H. Wang*, D.H.H. Tsao**, J. Ferretti**, R. Chmelik*, and M. Nirenberg*; Laboratory of Biochemical Genetics* and Laboratory
of Biophysical Chemistry**, NIH, Bethesda, Maryland.
The NK-2 homeobox gene (Kim,Y., and Nirenberg, M. (1989) Proc. Natl. Acad. Sci. USA, 86,77l6-7720) is expressed initially
by nuclei in late stage 4 or early stage 5 Drosophila embryos that comprise the ventral half of the ventral neurogenic anlage
and by some nuclei in the procephalic region. These nuclei are precursors of neuroectodermal cells that give rise to subsets
of neuroblasts, ganglion mother cells, and neurons in the subesophageal ganglion, ventral nerve cord, stomatogastric nervous
system, and some cephalic ganglia. NK-2 also is expressed in the anterior and posterior midgut primordia. (Nakayama, K., Nakayama,
N. Kim, Y., Webber, K., Lad, R., and Nirenberg, M., in preparation). The NK-2 homeodomain and flanking regions (77 amino
acid residues, termed NK-2H) was synthesized in E. coli and purified. Nucleotide sequences of DNA binding sites for NK-2H
were identified by purifying oligonucleotides with central random sequences by repetitive NK-2H-Sepharose affinity column
chromatography. Purified oligonucleotides were amplified by PCR and cloned. The consensus nucleotide sequence of the NK-2H
DNA binding site, obtained by sequencing 77 clones, is T(T/C)AAGTG(G/C). The KD of NK-2H for the consensus sequence is 2 x
10^(-l0) M. Twenty putative high-affinity and 13 lower affinity NK-2 binding sites were found in 2.2 kb of the 5'-flanking
sequence of the NK-2 gene, which suggests that NK-2 protein may be required to maintain NK-2 gene expression. Many NK-2 binding
sites for DNA were found with overlapping or adjacent putative sites for other proteins, such as E(spl)m8, dorsal, snail,
or other homeodomain proteins, which suggests that NK-2 protein may compete with some proteins that regulate gene expression
for occupancy of NK-2 binding sites in DNA
Circular dichroism measurements and 1D NMR spectra showed that the tm for denaturation of NK-2H is approximately 25 degrees
C at pH 4.4 and that denaturation is fully reversible. NK-2H has considerably less alpha-helical content and a less stable
conformation than does the Antp homeodomain (Otting, G. et al. (1989) EMBO J. 7, 4305-4309). NK-2H uniformly enriched with
15N was examined by 2D and 3D NMR. The results show that NK-2H has a novel homeodomain conformation.