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The Marshall W. Nirenberg Papers

Recognition of DNA by the NK-2 Homeodomain pdf (91,629 Bytes) transcript of pdf
Recognition of DNA by the NK-2 Homeodomain
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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1 (91,629 Bytes)
1997-07-28 (July 28, 1997)
Wang, Lan-Hsiang
Tsao, D. H.H.
Ferretti, J.
Chmelik, Rebecca
Nirenberg, Marshall W.
This item is in the public domain. It may be used without permission.
Exhibit Category:
From Neuroblastoma to Homeobox Genes, 1976-1992
Box Number: 7
Folder Number: 6
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Document Type:
Abstracts (summaries)
Physical Condition:
Series: Series III: Laboratory Administration, [1959]-1993
SubSeries: Annual Reports, 1960-1993
Folder: XXXXXXXXXX, 1988-1993
Neurobiology of Drosophila
Lan-Hsiang Wang
Recognition of DNA by the NK-2 Homeodomain
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.
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