Molecular and Cellular Endocrinology, 82 (1991) 275-283 0 1991 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/91/$03.50
MOLCEL
275
02656
A preproinsulin-like
from Neurospora crassa
pseudogene
Ganapathy Muthukumar
and John Lenard
Department of Physiology and Biophysics, UMDNJ, Robert Wood Johnson Medical School (at Rutgers), Piscataway, NJ 0#54-5635, U.S.A. (Received
Key words: Neurospora crassa; Insulin;
Pseudogene;
1 August
1991; accepted
Polymerase
30 August
1991)
chain reaction
Summary A segment of DNA was amplified from the Neurosporu crassa genome reaction using several oligonucleotides coding for highly conserved domains in probe. A genomic clone corresponding to this segment was isolated and the determined. The deduced amino acid sequence of a part of this segment bears to preproinsulin, but lacks several requirements for transcription or translation considered to be a pseudogene.
Introduction Previous work from this laboratory has provided evidence for a functional insulin-dependent signal transduction pathway in Neurospora crassa cells. A specific insulin-binding plasma membrane ‘receptor’ was isolated and purified to homogeneity (Kale, 1991). Further, the addition of mammalian insulin to these cells produced numerous metabolic effects, including the following: enhanced rates of production of CO,, ethanol, alanine and glycogen from glucose (Fawell et al., 1988; Greenfield et al., 1988, 1990; MacKenzie et al., 1988); activation of the enzyme glycogen synthase from a glucose-6-phosphate dependent to
Address for correspondence: G. Muthukumar, Department of Physiology and Biophysics, UMDNJ, Robert Wood Johnson Medical School fat Rutgers), 675 Hoes Lane, Piscataway, NJ 08854-5635, U.S.A. Tel. (908) 463-4784; Fax (908) 463-5038.
by the polymerase chain proinsulin as primers and nucleotide sequence was remarkable resemblance and must therefore be
an independent form (Fawell et al., 19881; phosphorylation of discrete cellular proteins, at serine, threonine and tyrosine residues (Kale and Lenard, 1991). Insulin-like proteins have been found in a wide variety of insects, invertebrates, plants, and microbial eukaryotes and prokaryotes (LeRoith et al., 1980, 1985~; Rubinovitz and Shiloach, 1985; Collier et al., 1987; Conlon et al., 1988; De Pablo et al., 1988; Maier et al., 1988; Robitzki et al., 19891, including N. crassa (LeRoith et al., 1980). Identification of insulin-like proteins in these organisms was made using the following criteria: cross-reactivity with anti-insulin antibodies; chromatographic characteristics resembling those of mammalian insulin on one or more separating systems; insulin-like bioactivity when tested on mammalian cells; neutralization of the bioactivity by anti-insulin antibodies, and/or by treatment of the target cells with anti-insulin receptor antibody. Although genes coding for insulin-like proteins have been reported from several inverte-
276
brates (Smit et al., 1985; Rubinovitz and Shiloach, 1985; Kawakami et al., 1989; Robitzki, 1989) none has yet been found in any microorganism. In light of the above, the isolation of an insulin gene from A? CMSSUwould be of obvious interest. We have sought such a gene extensively, by screening various cDNA libraries with consensus oligonucleotide probes and anti-insulin antibodies; these efforts have been unsuccessful (S. Fawell, M. Hussain, G. Muthukumar, unpublished observations). We now describe a segment of DNA isolated from the N. crassa genome using polymerase chain reaction (PCR) (Saiki et al., 1988). The deduced protein sequence of this segment bears remarkable resemblance to preproinsulin, but lacks several requirements for transcription or translation, and must therefore be considered to be a pseudogene. Several features of this sequence, however, including the presence of a degenerate intron, suggest that it derives from an authentic N. crassa gene, and not from a recent recombinational event. Materials
and methods
Restriction enzymes and DNA modifying enzymes were obtained from BRL. Radiolabelled materials were purchased from NEN and ICN. Sequenase was purchased from USB. All other reagents were purchased from Sigma Chemical co. Strains N. crussu strain FGSC 4761 was obtained from
Fungal Genetic Stock Center, Arcata, CA, U.S.A. It is a rapidly growing variant of the wall-less slime mutant 1118 (Scarborough, 1985). Escherichia coli strains DHSaF’ and XL-1 Blue were used. Subclones were constructed in Ml3 and pUC vectors (Yanisch-Peron et al., 1985). Nucleic acid preparation
Genomic DNA was prepared as described by Sherman et al. (1981). Plasmid DNA was purified by the polyethylene glycol precipitation method. Total RNA and poly(A)+ RNA were prepared as described by Berlin and Yanofsky (1985). Radiolabelled DNA probes were prepared by random priming (Feinberg and Vogelstein, 1983). South-
ern and Northern analysis were done as per standard methods (Sambrook et al., 1989). Oligonucleotide primers /probes
Oligonucleotides were synthesized on an Applied Biosystems Model 380B DNA synthesizer. ‘Forward’ and ‘reverse’ primers were designed as coding sequences for the conserved insulin regions CGSHL and LENYCN, respectively, taking into consideration the frequency of codon usage in N. crassa. PCR products obtained using these primers were analyzed further using an ‘internal’ probe, directed against sequences coding for the conserved insulin region RGFFY. The forward and reverse primer sequences were appended to an EcoRI and a BumHI restriction site, respectively. The sequences were as follows: forward
5’-CTAGAATTCTG(T/
C)GGNTCNCA(T/C)
(T/C)T-3’ reverse
5’-CCGGGATCC(G/A)TT(G/A)CAoTA
internal
5’-(G/A)TA(G/A)AA(G/A)AANCCNCG-3’
MOLCEL
275
02656
A preproinsulin-like
from Neurospora crassa
pseudogene
Ganapathy Muthukumar
and John Lenard
Department of Physiology and Biophysics, UMDNJ, Robert Wood Johnson Medical School (at Rutgers), Piscataway, NJ 0#54-5635, U.S.A. (Received
Key words: Neurospora crassa; Insulin;
Pseudogene;
1 August
1991; accepted
Polymerase
30 August
1991)
chain reaction
Summary A segment of DNA was amplified from the Neurosporu crassa genome reaction using several oligonucleotides coding for highly conserved domains in probe. A genomic clone corresponding to this segment was isolated and the determined. The deduced amino acid sequence of a part of this segment bears to preproinsulin, but lacks several requirements for transcription or translation considered to be a pseudogene.
Introduction Previous work from this laboratory has provided evidence for a functional insulin-dependent signal transduction pathway in Neurospora crassa cells. A specific insulin-binding plasma membrane ‘receptor’ was isolated and purified to homogeneity (Kale, 1991). Further, the addition of mammalian insulin to these cells produced numerous metabolic effects, including the following: enhanced rates of production of CO,, ethanol, alanine and glycogen from glucose (Fawell et al., 1988; Greenfield et al., 1988, 1990; MacKenzie et al., 1988); activation of the enzyme glycogen synthase from a glucose-6-phosphate dependent to
Address for correspondence: G. Muthukumar, Department of Physiology and Biophysics, UMDNJ, Robert Wood Johnson Medical School fat Rutgers), 675 Hoes Lane, Piscataway, NJ 08854-5635, U.S.A. Tel. (908) 463-4784; Fax (908) 463-5038.
by the polymerase chain proinsulin as primers and nucleotide sequence was remarkable resemblance and must therefore be
an independent form (Fawell et al., 19881; phosphorylation of discrete cellular proteins, at serine, threonine and tyrosine residues (Kale and Lenard, 1991). Insulin-like proteins have been found in a wide variety of insects, invertebrates, plants, and microbial eukaryotes and prokaryotes (LeRoith et al., 1980, 1985~; Rubinovitz and Shiloach, 1985; Collier et al., 1987; Conlon et al., 1988; De Pablo et al., 1988; Maier et al., 1988; Robitzki et al., 19891, including N. crassa (LeRoith et al., 1980). Identification of insulin-like proteins in these organisms was made using the following criteria: cross-reactivity with anti-insulin antibodies; chromatographic characteristics resembling those of mammalian insulin on one or more separating systems; insulin-like bioactivity when tested on mammalian cells; neutralization of the bioactivity by anti-insulin antibodies, and/or by treatment of the target cells with anti-insulin receptor antibody. Although genes coding for insulin-like proteins have been reported from several inverte-
276
brates (Smit et al., 1985; Rubinovitz and Shiloach, 1985; Kawakami et al., 1989; Robitzki, 1989) none has yet been found in any microorganism. In light of the above, the isolation of an insulin gene from A? CMSSUwould be of obvious interest. We have sought such a gene extensively, by screening various cDNA libraries with consensus oligonucleotide probes and anti-insulin antibodies; these efforts have been unsuccessful (S. Fawell, M. Hussain, G. Muthukumar, unpublished observations). We now describe a segment of DNA isolated from the N. crassa genome using polymerase chain reaction (PCR) (Saiki et al., 1988). The deduced protein sequence of this segment bears remarkable resemblance to preproinsulin, but lacks several requirements for transcription or translation, and must therefore be considered to be a pseudogene. Several features of this sequence, however, including the presence of a degenerate intron, suggest that it derives from an authentic N. crassa gene, and not from a recent recombinational event. Materials
and methods
Restriction enzymes and DNA modifying enzymes were obtained from BRL. Radiolabelled materials were purchased from NEN and ICN. Sequenase was purchased from USB. All other reagents were purchased from Sigma Chemical co. Strains N. crussu strain FGSC 4761 was obtained from
Fungal Genetic Stock Center, Arcata, CA, U.S.A. It is a rapidly growing variant of the wall-less slime mutant 1118 (Scarborough, 1985). Escherichia coli strains DHSaF’ and XL-1 Blue were used. Subclones were constructed in Ml3 and pUC vectors (Yanisch-Peron et al., 1985). Nucleic acid preparation
Genomic DNA was prepared as described by Sherman et al. (1981). Plasmid DNA was purified by the polyethylene glycol precipitation method. Total RNA and poly(A)+ RNA were prepared as described by Berlin and Yanofsky (1985). Radiolabelled DNA probes were prepared by random priming (Feinberg and Vogelstein, 1983). South-
ern and Northern analysis were done as per standard methods (Sambrook et al., 1989). Oligonucleotide primers /probes
Oligonucleotides were synthesized on an Applied Biosystems Model 380B DNA synthesizer. ‘Forward’ and ‘reverse’ primers were designed as coding sequences for the conserved insulin regions CGSHL and LENYCN, respectively, taking into consideration the frequency of codon usage in N. crassa. PCR products obtained using these primers were analyzed further using an ‘internal’ probe, directed against sequences coding for the conserved insulin region RGFFY. The forward and reverse primer sequences were appended to an EcoRI and a BumHI restriction site, respectively. The sequences were as follows: forward
5’-CTAGAATTCTG(T/
C)GGNTCNCA(T/C)
(T/C)T-3’ reverse
5’-CCGGGATCC(G/A)TT(G/A)CAoTA
internal
5’-(G/A)TA(G/A)AA(G/A)AANCCNCG-3’
