Structure and expression of the chicken ,3 nerve growth factor

The EMBO Journal vol.5 no.7 pp.1483-1487, 1986

Structure and expression of the chicken ,3

nerve

growth factor

gene

Ted Ebendal, Dan Larhammarl and H.akan Persson1 Departments of Zoology and 'Medical Genetics, Uppsala University, S-751 22 Uppsala, Sweden Communicated by U.Pettersson

The 3' exon of the chicken ,3 nerve growth factor (NGF) gene was isolated by the use of a murine cDNA probe. DNA sequence analysis of the clone suggests a mature chicken NGF protein of 118 amino acids, showing -85% homology to mouse and human NGF. In addition to this conservation of the mature NGF, parts of the propeptide and the untranslated 3' end of the NGF gene are also highly homolgous in chicken, human and mouse. Therefore, these sequences probably subserve important functions. Expression of NGF mRNA in various chicken tissues was examined by RNA blot analysis with a chicken NGF probe. A single mRNA of 1.3 kb was detected at high levels in heart and brain of 10-week-old roosters, and, at lower levels in spleen, liver and skeletal muscle. These data suggest a correlation between NGF expression and the density of sympathetic innervation in peripheral organs, in analogy with rmdings for mammalian tissues. In the adult avian brian, NGF mRNA is found at higher concentration in the optic tectum and cerebellum than in the cortex and hippocampus. This pattern of NGF expression differs from that previously described for the rat brain. During late stages of development (day 18), NGF mRNA was expressed both in heart and brain of embryos but at lower levels than in the adult. Key words: brain/chicken embryo/NGF protein sequence/RNA blot

Introduction ,3 nerve growth factor (NGF) is a protein of 118 amino acids that functions as a trophic factor for sympathetic and sensory neurons (Levi-Montalcini and Angeletti, 1968; Thoenen and Barde, 1980). Murine cDNA clones for NGF have been described (Scott et al., 1983; Ullrich et al., 1983) and were used to isolate the human NGF gene (Ullrich et al., 1983). DNA sequence analysis showed that the major part of the prepro-NGF is encoded in one exon. In addition, the NGF gene consists of at least two small 5' exons, including an alternative initiation site for translation (Ullrich et al., 1983; Edwards et al., 1986). Recently, RNA blot analysis and enzyme immunoassay were used to demonstrate the presence of NGF mRNA and protein in subregions of the mammalian brain (Korsching et al., 1985; Whittemore et al., 1986), where NGF has been suggested to regulate cholinergic function (Gnahn et al., 1983; Mobley et al., 1985; Korsching et al., 1985). In contrast to the well-documented presence of NGF in mammals (Heumann et al., 1984; Shelton and Reichardt, 1984), little is known about the presence or function of NGF in avian tissues. Developing chicken sensory and sympathetic neurons have the high-affinity NGF receptor (Sutter et al., 1979; Rohrer © IRL Press Limited, Oxford, England

et al., 1983; Raivich et al., 1985), show retrograde axonal transport of injected murine NGF (Brunso-Bechtold and Hamburger, 1979) and respond with increased survival, cell size and neurite growth to exogenous NGF (Levi-Montalcini and Hamburger, 1953; Cohen et al., 1954). Evidence for a chicken NGF protein has also been obtained from measurements of biological NGF-like activity in conditioned medium from chicken cells (Young et al., 1975; Norrgren and Ebendal, 1986) and explanted chicken irides (Ebendal et al., 1982). More recently, antibodies to mouse ,BNGF were shown by radioimmunoassay and by immunoblot to recognize a chicken embryo protein of mol. wt 13 000 daltons (Ebendal et al., 1984; Belew and Ebendal, 1986). In order to deduce the structure of the chicken NGF polypeptide, we screened a chicken genomic phage library with a murine cDNA probe (Scott et al., 1983). We describe here one clone which is shown by sequence analysis to encode a protein highly homologous to mammalian NGF. Using a probe from this gene, levels of NGF mRNA were determined in adult and embryonic chicken tissues, including the brain.

Results Characterization of the chicken prepro-NGF gene A chicken genomic library in Charon 28 was screened with a 900-bp PstI fragment derived from a murine (3NGF cDNA clone (Scott et al., 1983). From a primary screen of 550 000 clones, one clone was isolated under stringent hybridization and washing conditions. A 2.4-kb Bgmll-BamHI fragment that hybridized to the murine NGF cDNA clone was subcloned in pUC 19 and subjected to DNA sequence determination (Figure 1). The nucleotide sequence and the predicted amino acid sequence are shown in Figure 2. The phage insert begins 136 bp 5' to the last exon of the NGF precursor gene. This exon encodes the carboxy-terminal 243 amino acids of prepro-NGF, including the mature NGF polypeptide, as well as the 3'-untranslated region of the NGF mRNA, in analogy with the corresponding exon in the human NGF gene (Ullrich et al., 1983). (BgI 11) Pst I

X

Bst Ell Nco I

I (-

1

Bam Hi

Pst I

I

.'5 Q

0

Pst I

I

1.0o

1.5

2.0

,

Fig. 1. Restriction map of chromosomal segment containing the last exon of the chicken prepro-NGF gene. The exon is shown as an open box. The 5' end of the exon is close to one arm of the bacteriophage X vector. The 2.4-kb BglII-BamHI fragment was subcloned in pUC19 and subjected to nucleotide sequence determination with the Maxam and Gilbert (1980) procedure, as indicated by arrows. Horizontal bar shows the 910-bp Pstl fragment used in hybridizations to mRNA and genomic DNA.

1483

T.Ebendal, D.Larhammar and H.Persson GATCAACATCTGGTACCCACATGAGAACCTGATGAAACCTGTATGGTCCTGGGTATCTTACACATTTATTGCCAGCAM CACCATCTGTTTCTGCCAGCGTGGCTTACAGTAATACTACAT

120

-125 Val His Ser Val Met Ser Met Leu Tyr Tyr Thr Leu Ile Ile Ala Phe Leu Ile Gly Thr Gln Ala Ala Pro Lys Ser TTTTCTITGTSTCTAG GTG CAT AGC GTA ATG TCC ATG CTG TAC TAC ACT CTG ATT ATA GCT mT] TTG ATC GGC ACA CAG GCA GCT CCA AAG TCA

-100 214

Glu Asp Asn Gly Pro Leu Glu Tyr Pro Ala Glu His Ser Leu Pro Ser Thr Gln Gln Ser Asn Gly Gln His Ile Ala Lys Ala Ala Pro GAG GAC AAT GGT CCG CTG GAG TAT CCT GCA GAA CAC TCC TTA CCC AGT ACA CAA CAG AGT AAC GGA CAG CAC ATT GCC AAG GCA GCC CCA

-70 304

Gln Thr Thr His Gly Arg Phe Ala Trp Met Pro Asp Gly Thr Glu Asp Leu Asn Ile Ala Met Asp Gln Asn Phe Phe ys Lys Lys Arg CAG ACA ACC CAC GGC CGC TTT GCT TGG ATG CCA GAT GGA ACA GAA GAT TTA AAT ATC GCT ATG GAC CAA AAC TTC TTT AAG AG AAA CGTJ

-40 394

Phe Ara Ser Ser Arg Val Leu Phe Ser Thr Gln Pro Pro Pro Val Ser Arg Lys Gly Gln Ser Thr Gly Phe Leu Ser Ser Ala Val Ser TTC CGG TCT TCT CGG GTC CTG TTC AGC ACG CAG CCA CCT CCA GTG TCA AGG AAA GGA CAG AGT ACG GGG TTT CTC AGC AGT GCA GTC TCT

-10 484

+1

Leu Asn Arg Thr Ala Arg Thr Lys Arg Thr Ala His Pro Val Leu His Arg Gly Glu Phe Ser Val Cys Asp Ser Val Ser Met Trp Val CTC AAC AGG ACT GCC AGG ACC AAG AGG ACT GCA CAT CCC GTA CTG CAC CGG GGA GAG TTC TCA GTG TGT GAC AGT GTC AGC ATG TGG GTC

21 574

Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Thr Val Leu Gly Glu Val Asn Ile Asn Asn Asn Val Phe Lys Gln Tyr GGG GAC AAA ACC ACC GCC ACC GAC ATC AAA GGC AAA GAG GTG ACC GTG CTG GGA GAG GTC AAC ATT MAC AAC MAC GTT m'I AAG CAG TAC

51 664

Phe Phe Glu Thr Lys Cys Arg Asp Pro Arg Pro Val Ser Ser Gly Cys Arg Gly Ile Asp Ala Lys His Trp Asn Ser Tyr Cys Thr Thr TTT TTC GAG ACC MAG TGC AGG GAC CCT AGG CCG GTG TCC AGC GGG TGC CGA GGG ATT GAT GCG MAG CAT TGG AAC TCT TAC TGC ACC ACG

81 754

Thr His Thr Phe Val Lys Ala Leu Thr Met Glu Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu ACA CAC ACC TTC GTC AAA GCA CTG ACC ATG GAG GGC MAG CAM GCA GCC TGG AGA TTT ATC CGG ATC GAC ACA GCC TGT GTG TGT GTG CTC

ill 844

Ser Arg Lys Ser Gly Arg Pro *A* AGC AGG AAA TCG GGG AGA CCC TGA GGCGGAGCTGAMCCCCACAMCAMCATCCTCCTATCCCCCTACCTCAGCCTGTAMTTATT MGITAAAAGAAAAAAAAAAATMGG

118 956

ACTGCATGGTGTATTTATAGTTTATACAMATACAAAGAGAMACCTCATTATT ATAAACTCT m

ACCTTTCGTGTTTTGCAMATTCATTGACCAGCTGGACTCTTCAAAAGWGTATTC

ACCTTCATAGGGATAMTGCAGCCCTACAGCTCTGAGCTGTTCATAGAGCTGCTGAGCAMTCTTTGTCACTCTTCTGCAG

1076

1156

Fig. 2. Nucleotide sequence and deduced amino acid sequence of the 3' exon of the chicken prepro-NGF gene. The sequence of mature NGF is underlined. Arrow indicates exon boundary. The presumptive polyadenylation signal is underlined. A potential glycosylation site is overlined by double lines. Potential proteolytic processing sites are within boxes.

A -187 MOUSE HUMAN CHICKEN

-180

-170

,

-160

-150

-140

-120

-130

-119

***

***********

S

S A

NS T G

P HT Y -125 -120

-70

-80

MOUSE HUMAN CHICKEN

MOUSE HUMAN CHICKEN

MOUSE HUMAN CHICKEN

-60

-50

-40

V L A A A SLPSTQ SNGQHIAKA PQTTHGRF WMPD TEDL

-80

-70

-90

-20

-10

HSE A HTI Q A KSED G LEYPAEH -lo -90

20

30

REAA V

R K FRS

AM QNF

-60

-50

-40

49

5p

160

Q

R39STG -20

-30

F79

89

EVG AA LSS*AVSL

-l

+1

10

K STHPVFHMGEF S I R A I4_ A* L R l 1

90

100

110

SVCDSVSVWVGDKTTATDIKGKEVTVLAEVNINNSVFkQYFFETKCRASNPVESGCRG,IDSKHWNSYCTTTHTFVKALTTDEKQAAWRZFIRIDTACVCVI M M 20

SRKA

30

G G 40

N

K K 50

DP DPR

66

D S

L

M G MEG

A 70

80

90

100

80

90

110

120 G

P

SG *P 118

10

20

30

50

40

60

70

10l

TFG*TTGCCtGCAGCCCCCtTCCCCACCT6CCCCCTCCAtACTCTCTTG6GCCCCTCCCtACCTCAGCC'TGTAAATTAT=AAATTA*AA********A A GA CG T C C T*TT C

LGGC GAGCTGAM 110

MOUSE HUMAN CHICKEN

-30

I T

I -110

AHWTKLQHSLDTA1f-SAPTAPIAARVTGQTRA*TDPRLF KKRiILHSPRVLFSTQPPPTSSDTLDLDFQAHGTIPFNRTHR

B MOUSE HUMAN CHICKEN

-100

MLCLKPVKLGSLEVGHGQHGGVLACGRAVQGAGWHAGPKLTSVSGPNKGFAKDAAFYTGRSEVHSVMSMLFYTLITAFLIGVQAEPYTDSNVPEGDSVPE

ACAA A A C T TAT*** A***********Ak C

120

130

150

140

G

160

170

kkkTAAGGACTGCATGATAAATTTATCGTTTATACAATTTTAA*AGAAAA*CA TAmTAAATTrC G G A A T AAA

G GT

A

ACA

G

ACCT

CTC

189

AAAGAAAAAAAA 190

C*AAAGCATCCTGAAAAAAA TGTGCTG TGG TGG A C TTC

TGITITGC

Fig.

3. (A) Alignment of prepro-NGF amino acid sequences from mouse, man and chicken. The mature NGF protein starts at position + 1. Arrows mark boundaries. Stars show gaps introduced to optimize homologies. Sequence information is not yet available for the first exons of the human and the chicken gene. Potential glycosylation sites are overlined by double lines. Potential proteolytic processing sites are within boxes. Numbering above the sequences refers to the murine and human sequences, whereas the numbering below the sequences refers to the chicken sequence. Cysteine residues likely to form disulfide bonds are connected by lines (Angeletti et al., 1973). (B) Alignment of the 3'-untranslated regions of the prepro-NGF genes of mouse, man and chicken. The termination codon of the protein translation is within a box, as is the potential polyadenylation signal. Arrow marks the poly(A) addition site in the murine sequence. Stars indicate gaps introduced to optimize homologies. exon

The mature NGF protein is remarkably well conserved (Figure 3A). The chicken sequence is 88% and 84% homologous to the human and murine sequences, respectively (the latter two are 90% homologous). The amino acid replacements are primarily

1484

located in the amino-terminal part, the carboxy-terminal part, and the small segment bounded by the disulfide bridges involving Cys-57 and Cys-67. At both termini of the mature NGF, the chicken polypeptide lacks one amino acid residue as compared

'

Chicken (3NGF gene sequence and expression

C,

CNe

C,e

I.P

V.

11