Sequence characteristics within nuclear genes from Sordaria macrospora

Stefanie Pöggeler- Ruhr-Universität, 44780 Bochum, Germany

This paper reports sequence features within nuclear genes from Sordaria macrospora. Eight nuclear gene sequences were analyzed for codon usage, GC content, intron regulatory sequences and translation initiation sites.

The homothallic ascomycete Sordaria macrospora is an excellent model system to study not only meiotic pairing and recombination (Zickler 1977 Chromosoma 61:29-316) but also fruiting body development (Esser and Straub 1958 Z. Vererbungslehre 89:729-746). Recently, these studies have been extended to a molecular level (Walz and Kück 1995 Curr. Genet. 29:88-95) and knowledge about sequence features would be a helpful tool in sequence analysis. Until now, sequence information from S. macrospora was only available from a single nuclear gene (LeChevanton and Leblon 1989 Gene 77:39-49). Here we compile sequence data from eight recently sequenced genes to determine common features of nuclear genes from S. macrospora.
We provide a consensus sequence for the translation initiation site (Table 1), a codon usage table (Table 2), and consensus sequences for intron regulatory sequences (Table 3). Comparison of the data presented here with sequence features from the well studied ascomycete Neurospora crassa (data taken from Brucherez et al. 1993 Fungal Genet. Newsl. 40:85-95; and Edelman and Staben 1994 Exp Mycol 18:70-81) shows that S. macrospora sequence characteristics are very similar to those determined for N. crassa genes.

Table 1. Translation initiation context
Gene Gene product Translation initiation Referencea
EF1- EF1- translation elongation factor CCGTCAAAATGGG 1
tuba -tubulin CATACAAAATGCG 2
ura3 orotidine phosphoribosyl transferase CCGCCACAATGTC 3
ura5 orotidine monophosphate decarboxylase CCAGCACAATGGC 4
SmtA-1 mating-type protein GAAGTACGATGTC 5
SmtA-2 mating-type protein CGACTGACATGGA 5
SmtA-3 mating-type protein CTTTCAGCATGTC 6
Smta-1 mating-type protein TCGAAACAATGGA 5

a (1)Gagny, Koll and Silar, unpublished (Accession # X96615) (2) Pöggeler et al., submitted (Accession # Z70290) (3) Nouwrousian, unpublished (Accession # Z70291) (4) Nouwrousian, unpublished (5) Pöggeler et al., submitted (Accession # Y10616) (6) Pöggeler, unpublished

Consensus translation initiation

S. macrospora 

		         A38              C50                 G50


		 C75* C50      N   C63  A88     A63  A100 T100 G100     C50


		         G38              A38                 T38


N. crassa


		                         A                  


		 C   N   N   N   C   A      A   A   T   G   G   C


		                         C
*The subscript number indicates the percentage occurrence of the particular nucleotide.

The S. macrospora consensus for initiation of translation shows a high degree of identity to the N. crassa translation initiation consensus sequence and, as N. crassa, a prevalence of GC following the ATG which means that an alanine (GCN) is found at the amino terminus of most proteins studied so far.

Table 2. Codon usage analysis based upon 2497 codons 

TTT-Phe  24( 25.0%)a     TCT-Ser  22( 16.4%)	TAT-Tyr  21( 26.9%)	TGT-Cys   3(8.6%)
TTC-Phe  72( 75.0%)	TCC-Ser  42( 31.3%)	TAC-Tyr  57( 73.1%)	TGC-Cys  32( 91.4%)
TTA-Leu   3(  1.7%)	TCA-Ser  12(  9.0%)	TAA-Ter   4( 57.1%)	TGA-Ter   1( 14.3%)
TTG-Leu  21( 11.6%)	TCG-Ser  28( 20.9%)	TAG-Ter   2( 28.6%)	TGG-Trp  32(100.0%)
CTT-Leu  45( 24.9%)	CCT-Pro  41( 30.6%)	CAT-His  23( 30.7%)	CGT-Arg  34( 27.6%)
CTC-Leu  80( 44.2%)	CCC-Pro  68( 50.7%)	CAC-His  52( 69.3%)	CGC-Arg  57( 46.3%)
CTA-Leu   3(  1.7%)	CCA-Pro  13(  9.7%)	CAA-Gln  24( 23.8%)	CGA-Arg   7(  5.7%)
CTG-Leu  29( 16.0%)	CCG-Pro  12(  9.0%)	CAG-Gln  77( 76.2%)	CGG-Arg   6(  4.9%)
ATT-Ile  47( 32.6%)	ACT-Thr  25( 19.2%)	AAT-Asn  17( 17.3%)	AGT-Ser   5(  3.7%)
ATC-Ile  95( 66.0%)	ACC-Thr  71( 54.6%)	AAC-Asn  81( 82.7%)	AGC-Ser  25( 18.7%)
ATA-Ile   2(  1.4%)	ACA-Thr  15( 11.5%)	AAA-Lys  12(  7.2%)	AGA-Arg   7(  5.7%)
ATG-Met  63(100.0%)	ACG-Thr  19( 14.6%)	AAG-Lys 154( 92.8%)	AGG-Arg  12(  9.8%)
GTT-Val  40( 24.2%)	GCT-Ala  70( 30.7%)	GAT-Asp  61( 42.4%)	GGT-Gly  61( 32.3%)
GTC-Val 101( 61.2%)	GCC-Ala 115( 50.4%)	GAC-Asp  83( 57.6%)	GGC-Gly  94( 49.7%)
GTA-Val   5(  3.0%)	GCA-Ala  21(  9.2%)	GAA-Glu  33( 19.0%)	GGA-Gly  24( 12.7%)
GTG-Val  19( 11.5%)	GCG-Ala  22(  9.6%)	GAG-Glu 141( 81.0%)	GGG-Gly  10(  5.3%)
aThe percent shown by each codon represents the percent of the time that the amino acid is encoded by that codon.

The GC content in a coding region of 7491 nucleotides is 56.7%. For comparison in N. crassa the GC content is 58.6% in the coding region (GC content in total DNA 54.1%). In cases where amino acids are represented by more than one codon, S. macrospora, as many other organisms, does not use synonym codons equally (Table 2).

In S. macrospora, as in N. crassa, codons are preferred with a C in the third position and in four codon families the codon ending in T is usually preferred to those ending in A or G. The stop codon TAA is more frequently used than TAG or TGA, respectively. The six least used codons for S. macrospora are ATA (Ile), TTA (Leu), CTA (Leu), TGT (Cys), GTA (Val), and AGT (Ser). All of these six codons are belonging to low-usage codons in N. crassa as well. As reported by Zhang et al. (1991 Gene 105:61-67) in many organisms, low-usage codons are clearly avoided in abundant proteins and therefore may affect translation rates.

Table 3. Intron regulatory sequences and intron length
Intron 5' Intron

Donor

 Branch

Site

 Distance to

3' Splice-Site/ntb

 3' Intron

Acceptor

 Intron

Length / bp

SmtA-1/1a T^GTAAGT ACTGATT -19- TTCAG^ 58
SmtA-1/2a G^GTTAGT ACTCGTG -21- GGCAG^ 60
SmtA-2/1a G^GTAACA ACTGATG -14- GCCAG^ 57
SmtA-2/2a G^GTGAGT ACTGACA -12- GATAG^ 71
SmtA-2/3a T^GTAAGA ACTAATA -12- GACAG^ 47
SmtA-2/4a G^GTTTGC GCTAACA -16- GACAG^ 55
SmtA-3/1a C^GTGAGT ACTGACT -12- GTTAG^ 54
Smta-1/1a A^GTAAGT ACTGACC -15- TTTAG^ 53
Smta-1/2a T^GTAGGT ACTAACC -12- CTTAG^ 57
tuba/1 G^GTACGT GCTAACG -22- TCTAG^ 256
tuba/2 G^GTAGGT GCTAACC -15- ATTAG^ 149
tuba/3 G^GTAAGC GCTAACC -17- TACAG^ 80
tuba/4 G^GTACAT GCTTACA -18- CACAG^ 60
tuba/5 G^GTATGT ACTAACT -16- CTTAG^ 64
tuba/6 T^GTAAGT GCTAACT -14- CCTAG^ 57
ef1/1 G^GTAATG GCTAACG -14- AACAG^ 100
ef1/2 G^GTTAGT ACTGACT -15- AACAG^ 243
ef1/3 G^GTATGT GCTAACT -17- AAAAG^ 60
a positions of intron splice sites inferred from cDNA sequences
b distance between the C of the intron branch point and the G of the 3' intron acceptor

Consensus 5' Intron-Donor

S. macrospora		 G67^ G100  T100   A72   A61   G83   T72
N. crassa		 G^   G    T     A     A    G     T

Consensus Intron Branch Site 

                         A56             A56
S. macrospora			C100   T100       A94   C78   N
			 G44             G33

			 A              A         C
N. crassa			C    T        A         C
			 G              G         A
Consensus 3' Intron-Acceptor 
		 G33  A39  C56
S. macrospora	               A100  G100
		 A27  T39  T44

		 A   A    T
N. crassa		         A    G
		 T   T    C
In S. macrospora genes the intron length lies between 47 bp and 256 bp, the average length is 88 bp and the median length is 60 bp. Intron length in N. crassa ranges from 46 to 856 bp with a tendency toward 60 to 70 bp. Among the eight genes analyzed so far, two genes, ura3 and ura5, do not contain introns. In S. macrospora introns the distance from the C of the splice branch site to the G of the 3' splice site is between 12 nt and 22 nt. This distance varies in N. crassa from 14 to 30 nucleotides. The S. macrospora intron signals (5' donor site, intron branch site and 3' intron acceptor site) are very similar to the N. crassa intron consensus sequences.

Acknowledgments

I would like to thank Prof. Dr. U. Kück (Bochum) for his generous support, M. Nouwrousian for providing the unpublished sequence from the S. macrospora ura3 and ura5 genes, and D. Hahn for critical reading of the manuscript. This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Bonn-Bad Godesberg).

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