DNA Sequence-]. DNA Sequencing and Mapping, Vol. 3 , pp. 49-54 Reprints available directly from the publisher Photocopying permitted by license only

0 1992 Harwood Academic Publishers CmbH Printed in the United Kingdom

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Gene sequences of chicken BDNF and NT-3 PETER C. MAISONPIERRE*, LEONARD0 BELLUSCIO, JOANNE C. CONOVER and GEORGE D. YANCOPOULOS Mitochondrial DNA Downloaded from informahealthcare.com by TIB/UB Hannover on 01/03/15 For personal use only.

Regeneron Pharmaceuticals, Inc., 777 Old Saw M i l l River Road, Tarrytown, N.Y. 10591-6707

GenBank Accession Nos. M83377 a n d M83378

The respective amino acid sequences of mature brain-derived neurotrophic factor (BDNF) and of mature neurotrophin-3 (NT-3) are identical among mammals, making these among the structurally conserved factors known. Here we show that only a single conservative amino ac'id substitution distinguishes the chicken mature NT-3 protein from its mammalian counterpart. Chicken mature BDNF shows slightly more variation, differing from mammalian BDNF at several positions. We also note the presence of amino acid sequence motifs in the precursor protein sequences of chicken BDNF and NT-3 that are universally conserved among all known mammalian neurotrophin precursors and have been demonstrated to play a crucial role in promoting correct processing of the pro-proteins.

oxy-terminal half of the protein precursor. Additionally, the mature protein moieties of all neurotrophins have conserved six cysteine residues that, in biologically active NGF, has been shown to form three intrachain disulfide bonds. Radiolabeled DNAs spanning the -800 bp protein coding regions of rat BDNF and NT-3 (Maisonpierre et a/., 1991) were each found to detect only a single fragment on a genomic Southern blot of EcoRI-digested chicken DNA, suggesting that BDNF and NT-3 genes are represented only once in the chicken haploid genome (data not shown). We used these same rat probes to screen a chicken genomic DNA library prepared from Sau3A partial-digest chicken liver D N A cloned in the EMBL3-SPGfl7 bacteriophage vector (library purchased from Clontech Laboratories, Inc.). Out of approximately 1 . 2 ~ 1 0 chicken " genomic clones screened, one NT-3-hybridizing clone and one BDNF-hybridizing clone were isolated and subjected to further analysis. The chicken NT-3 D N A sequence was determined from a 3 kb Pstl fragment of the phage clone that hybridized with the rat NT-3 probe described above. This chicken genomic fragment was subcloned into pBluescript II (Stratagene, Inc.) and sequenced according to the manufacturer's protocols with the Sequenase, version 2, kit ( U S Biochemical, Inc.) and the dideoxynucleotide chain termination method (Sanger et a/., 1977). Oligonucleotide primers that were previously used to sequence rat and human NT-3 (Maisonpierre e t a / . , 1990, 1991), as well as primers specific to the chicken NT-3 gene, were used to determine the chicken NT-3 gene sequence.

KEY WORDS: BDNF, NT-3, chicken genes, neurotrophic factors, nerve growth factors

INTRODUCTION Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) belong to a family of small, basic, secreted proteins that includes nerve growth factor (NGF) and neurotrophin-4 (NT-4) (Hallbook eta/., 1991 ; Ip eta/., 1992). These proteins, collectively termed neurotrophiris, have been shown to be crucial for the development and maintenance of the vertebrate nervous system (see Snider and Johnson, 1989). Several structural features are shared among the neurotrophins. Each one i s synthesized as a larger protein precursor that i s subsequently cleaved to yield a mature protein of about 120 amino acids, corresponding to the carb-

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Mitochondrial DNA Downloaded from informahealthcare.com by TIB/UB Hannover on 01/03/15 For personal use only.

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P. C. MAISONPIEKRE ET AL.

rhr. chit I k c v t3DNF gene sequence was determined i n two ways Initial sequence data were obt,iincd using the BDNF phage clone D N A as a templdtc> anti oligonuc leotide primers that were previously used to sequence rat and h u m a n BDNF (Maisonpierit (71 a / , 1090, 1901 j. Phage D N A was sequc.nc etl with the Sequenase, version 2, kit using nwthods th,it were moditird nteti ‘inti t h e tidelity of the PCR-derived KilNF st’titwn( t x is confirmed by independent src4ur~ntin8 i u n s using vNt and NT respectively, aligned with previously reported DNA sequences from ?),

various mammals, pig and mouse BDNF ( D N A sequences are from Leibrock et a / (10891 and Hofer et d / (19901, respectivelv, mouse N l - 3 cDNA sequence i s trom Hohn et d / (1990), c I I N A and genomic sequence encoding rat and h u m a n BDNF ,ind NT-3 are from Maisonpierre t>f ,I/ (1 990, 1991), Rosenthal ef , I / (1990), K a i s h o t’t ‘ j / (1 990) and Ernfors e t a / (1 990) Like all mamniali;rn KDNF a n d NT-3 genes examined so far, the entire tocling region tor chicken prepro-BDNF and prepro-NT- 3 is t o n tained on a single exon However, i t should he noted that, as seen in other neurotrophin genes, the chicken NT-3 gene possesses ,in intron-cxon splice-acceptor motit just upstre,im ot the A T C translation initiation codon (SPLIC t 1 ) , me h,ive not determined whether this putsplit e site i s also present in the chicken KDNF gcnc Evidentc. from various neurotrophin genes (Ebenclc31 el , I / , 1986, Selby et ‘ i / , 1987, fdwartls et , I / , 1088, Maisonpierre et , I / , 1990, 1991 j indic‘ites that longer forms of protein precursor\ (

Gene sequences of chicken BDNF and NT-3.

The respective amino acid sequences of mature brain-derived neurotrophic factor (BDNF) and of mature neurotrophin-3 (NT-3) are identical among mammals...
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