SHORT COMMUNICATION

cDNA cloning and physical mapping of porcine 3b-hydroxysteroiddehydrogenase/D5-D4 isomerase

A. von Teichman*, H. Joerg*, P. Werner², B. Brenig³ and G. Stranzinger**Institute of Animal Science, Swiss Federal Institute of Technology, ETH Zentrum, Zurich, Switzerland. ²St Luke's-Roosevelt Hospital Center,

New York, USA. ³Institute of Veterinary Medicine, University of GoÈ ttingen, GoÈ ttingen, Germany

Summary The 3b-hydroxysteroid dehydrogenase/D5-D4-isomerase (3b-HSD) enzymes are

essential for the biosynthesis of steroid hormones. The 3b-HSD gene family has been

reported to encode for different isoenzymes which function either as dehydrogenase/

isomerase or as reductase. The 3b-HSD enzymes are involved in the formation of the

pheromone androstenone (5a-androst-16-ene-3-one) which contributes to the

unpleasant odour present in the meat of uncastrated boars. An reverse-transcription±

polymerase chain reaction (RT±PCR) probe from porcine testicular tissue of a 3b-HSD

enzyme was used to screen a porcine adipose tissue cDNA library. Both strands of the

positive clones were sequenced and the putative coding sequence of 1122 nucleotides

encodes 374 amino acids. Comparison of the putative open reading frame with the

bovine and the human type I homologues revealed 85.6 and 79.3% identity,

respectively. Fluorescence in situ hybridization (FISH) was performed with a labelled

PAC clone containing the gene of interest. The 3b-HSD gene was mapped to the

porcine chromosome 4q16-4q21 which is in accordance with the comparative gene

map.

Keywords 3b-hydroxysteroid dehydrogenase/isomerase, androstenone, boar taint,

porcine chromosome 4, steroidogenesis.

The 3b-hydroxysteroid dehydrogenase/D5-D4-isomerase

(3b-HSD) isoenzymes play an essential role in the biosyn-

thesis of all classes of hormonal steroids in classical ste-

roidogenic, as well as in peripheral tissues (Bain et al. 1991;

Lachance et al. 1991; Abbaszade et al. 1995). The isoen-

zymes catalyse the oxidative conversion of D5-3b-hydroxy-

steroids to the D4-3-keto con®guration and they also

function as reductase. The 3b-HSD is involved in the for-

mation of the pheromone androstenone (5a-androst-16-

ene-3-one) which contributes to the unpleasant odour

present in the meat from uncastrated boars, also known as

`boar taint'.

The structures of several cDNAs encoding 3b-HSD

isoenzymes have been characterized in both the human and

other vertebrate species. The isoenzyme types I and II are

found in humans (Luu-The et al. 1989; Lachance et al.

1990; Lorence et al. 1990; RheÂaume et al. 1991), isoen-

zyme types I, II, III, IV, V and VI in the mouse (Bain et al.

1991, 1993; Abbaszade et al. 1995, 1997), isoenzyme

types I, II, III and IV in the rat (Simard et al. 1991a, 1993)

and isoenzyme types I, II and III in the hamster (Rogerson

et al. 1995). Macaque (Simard et al. 1991b), bovine (Zhao

et al. 1989), equine (Boerboom & Sirois 1997), chicken

(Nakabayashi et al. 1995) and rainbow trout (Sakai et al.

1994) 3b-HSD genes have also been studied, but to date

only one gene has been cloned from these species.

Two functionally distinct groups have been reported

in the mouse. Types I, III, VI, and most likely type II

function as dehydrogenase (EC 1.1.1.145) and isomerase

(D5 ® D4, EC 5.3.3.1), whereas types IV and V only

function as reductase.

The different types of the 3b-HSD gene usually appear to

be expressed in an organ-speci®c manner. For example, the

human type I 3b-HSD has been detected in the placenta,

Address for correspondence

G. Stranzinger, Department of Animal Science, Swiss Federal Institute

of Technology, ETH Zentrum, 8092 Zurich, Switzerland.

E-mail: stranzinger@inw.agrl.ethz.ch

Accepted for publication 4 August 2000

Ó 2001 International Society for Animal Genetics, Animal Genetics, 32, 298±302

skin and mammary gland (Lorence et al. 1990; RheÂaume

et al. 1991), whereas type II is predominantly expressed in

the adrenals as well as the gonads (Lachance et al. 1991;

RheÂaume et al. 1991).

In order to characterize the enzyme or enzymes involved

in the production of androstenone, members of the porcine

3b-HSD isoenzymes will be characterized. For this purpose

total RNA was extracted from the testicular tissue of an

adult boar as described by Neuenschwander et al. (1996).

Primers (HSDhumF2 and HSDhumR4, Table 1), chosen

from exon 3 and exon 4, were designed based on the human

3b-HSD type I which shares high identity and similarity to

the human, bovine and equine 3b-HSD gene sequences.

Using the oligo-(dT) primer, reverse transcription (RT) was

performed in a ®nal reaction volume of 25 ll with 2.5 lg

total RNA extracted from porcine testis, 4 lM primer, 1 ´RT buffer, 10U Rnasin (Promega, Madison, WI, USA), 4 mM

Na-Pyrophosphate, 0.25 lM dNTPs and 10U AMV Reverse

Transcriptase (Promega, Madison, WI, USA). Conditions

were at 70 °C for 5 min, cooling to 25 °C in 10 min, 42 °C

for 45 min, 50 °C for 10 min, 55 °C for 10 min, and ®nally

70 °C for 15 min. This was followed by polymerase chain

reaction (PCR) in a ®nal reaction volume of 25 ll of

1 ´ PCR buffer containing 5 ll RT product, 0.4 lM HSD-

humF2, 0.4 lM HSDhumR4, 0.2 mM dNTPs and 2.5U Taq

DNA Polymerase (Amersham Pharmacia Biotech, Buck-

inghamshire, UK). The PCR pro®le was 95 °C for 5 min,

followed by 35 cycles of 95 °C for 30 s, 56 °C for 30 s and

72 °C for 30 s, and a ®nal extension of 72 °C for 7 min. The

PCR product had the expected size of 801 bp and was

sequenced to determine whether the obtained fragment

belonged to the gene of interest. The DNA sequencing was

performed by the dideoxy chain-termination method with

the ABI 377 DNA sequencer (Applied Biosystems, Perkin-

Elmer Corp., Foster City, CA, USA). Alignments were

performed using the National Center for Biotechnology

Information's (NCBI) Basic Local Alignment Search Tool

(BLAST) (Altschul et al. 1997). The obtained sequences

were compared with existing sequences of the 3b-HSD gene

of other species in the GenBank using the GCG sequence

analysis software package version 8.2 (Genetic Computer

Group, Madison, WI, USA). From these sequences it was

possible to create porcine speci®c primers (Table 1).

A porcine adipose tissue cDNA library (Werner et al.

1999) was screened with a probe obtained by reverse-

transcription±polymerase chain reaction (RT±PCR) from

porcine testis using primers HSDhumF2 and HSDpR1

(Table 1), which produces a fragment of 732 bp. The

RT±PCR product was labelled with [a-32P]dATP (Prime-ItÒ

II Random Primer Labeling Kit, Stratagene, La Jolla, CA,

USA) and hybridized to »500 000 phages of the cDNA

library by in situ plaque hybridization. Five clones were

obtained after the second screening. One of these clones

harboured the complete coding sequence of the 3b-HSD

gene (GenBank accession number: AF232699). The other

clones obtained from the screening were truncated lacking

the 5¢-end of the coding sequence. However, as the 3¢-ends

of all ®ve clones were identical, this would suggest that the

cDNA library contains only one member of the 3b-HSD

genes. The putative open reading frame (ORF) with a length

of 1122 nucleotides of this complete clone encodes a 374

amino acids protein with a calculated molecular mass of

41 851 Da. This is in agreement with the human 3b-HSD

type I which was reported to have a molecular mass of

42 216 Da (Luu-The et al. 1989) and the bovine 3b-HSD, a

molecular mass of 42 093 Da (Zhao et al. 1989). All other

types known have been found to contain 373 amino acids.

The porcine 3b-HSD coding sequence shares 85.6, 81.7,

79.3, and 79.5% identity and the deduced amino acid

sequence 88.5, 82, 79.3 and 79.2% similarity with the

nucleotide and amino acid sequence of bovine, equine,

human type I and human type II 3b-HSD cDNA, respect-

ively (Fig. 1). The amino acid sequences of the mouse

dehydrogenase/isomerase types I and VI share 76 and

75.9% similarity with the porcine amino acid sequence.

The reductase types IV and V share 71.9 and 71.8% amino

acid sequence similarity with that of the porcine amino acid

sequence. The comparison of the porcine amino acid

sequence to human types I and II revealed no signi®cant

differences, therefore it is not possible to determine the

corresponding type. However, because of the similarities

with the mouse dehydrogenase/isomerase types and

reductase types, the described porcine 3b-HSD clone

appears to function as a dehydrogenase/isomerase.

A porcine PAC library (Al-Bayati et al. 1999) was

screened by PCR with a porcine with the primers HSDpF1

Table 1 Primer sequences (synthesized by

Microsynth GmbH, Balgach, Switzerland).Primer Sequence

Nucleotide position

5' ® 3' Ta (°C)

HSDhumF2 5¢-TGTCAATGTGAAAGGTACCCAGC-3¢ 56

HSDhumR4 5¢-CACAAGGGAACCAACCCACTCC-3 56

HSDpF1 5¢-ACCTCCCCAAAGCTACGATAACC-3¢ 955 ® 974 58

HSDpR1 5¢-CAGATCTCGCTGCGCCTTCTTG-3¢ 1186 ® 1165 58

Ta, Annealing temperature.

Ó 2001 International Society for Animal Genetics, Animal Genetics, 32, 298±302

cDNA cloning and physical mapping 299

and HSDpR1 (Table 1). These primers amplify a fragment of

235 bp, which was sequenced to con®rm that the ampli®-

cation was speci®c. The obtained PAC clone (IVM-

PAC714D9A472) was digested with restriction enzyme

Sau3AI. The digested product was labelled with Biotin-16-

dUTP using the random priming DNA labelling method

with the Prime-ItÒ Fluor Fluorescence Labeling Kit (Strata-

gene, La Jolla, CA, USA). The labelled probe were hybridized

by in situ hybridization to porcine metaphases.

The chromosome metaphases were analysed and photo-

graphed with a 63´ oil objective using a ¯uorescence light

microscope, QuantixÔ-Camera (Photometrics, Tuscon, AZ,

USA) and electronically stored (Scanalytics, IPLab for

Windows, Version 2.311). These metaphases were

Figure 1 Comparison of the deduced amino acid sequences of the porcine, human types I and II, bovine and equine 3b-HSD. The dashes (±)

represent amino acids that are identical to the porcine 3b-HSD.

Figure 2 (a) Porcine RBA-stained metaphase. The arrow indicates the chromosome 4¢s, (b) the same metaphase after FISH with the PAC clone as

probe with the ¯uorescent signals on the q-arm of chromosome 4 (arrows).

Ó 2001 International Society for Animal Genetics, Animal Genetics, 32, 298±302

von Teichman, Joerg, Werner, Brenig, Stranzinger300

compared with the pictures of the same RBA-banded

metaphases. The porcine 3b-HSD gene was mapped to

chromosome 4q16-4q21 (Fig. 2). In the mouse, 3b-HSD

isoforms have been found to be closely linked to chromosome

3 (Bain et al. 1993). In man the two known types have also

been shown to be in close genetic linkage (Russell et al.

1994) and have been allocated to chromosome 1p13

(Morrison et al. 1991). The segment on mouse chromosome

3 containing the 3b-HSD genes shows conservation of gene

order with the corresponding region of human chromosome

1. The mapping of the porcine 3b-HSD gene to chromosome

4q16-4q21 is in agreement with the comparative gene map

(Rettenberger et al. 1995). Human chromosome 1, which

contains the 3b-HSD genes, was shown to correspond to the

evolutionarily conserved fragments located on porcine

chromosomes 4, 6, 9, 10 and 14. Porcine chromosome

4q16-24 contains the NGFB gene which lies at position p13

on human chromosome 1, the same position as the human

3b-HSD genes are located and supporting the presence of

the porcine 3b-HSD found in this position.

The different types of the 3b-HSD gene family appear to

be expressed in different organs, therefore various tissues

will be analysed to identify and characterize further mem-

bers of the 3b-HSD gene family.

Acknowledgements

We would like to thank David Betts for his critical review

and comments to the manuscript. This study was supported

by the ETH Zurich project-number 0-20693-99.

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