African Journal of Biotechnology Vol. 2 (7), pp. 185–188, July 2003   

ISSN 1684-5315  © 2003 Academic Journals

Full Length Research Paper

Analysis of AVR4 promoter by sequential response-element deletion                

Olukosi, YA¹ and Iwalokun, BA²*

¹Genetics Division, Nigerian Institute of Medical Research (NIMR), 6, Edmond Crescent, Yaba – Lagos, PMB 2013, Lagos - Nigeria.

*Corresponding author: E-mail: bamwal@yahoo.com

Accepted 24 June 2003

Several reports have associated the variability in physico-chemical properties of avidin protein to dynamism inherent in the consensus regulatory networks within the promoter region of avidin genes. An Avr4 promoter region ligated to chloramphenicol acetyltransferase plasmid vector (pBLCAT2) to produce recombinant plasmid Avr4pBLCAT2 was sequentially deleted to produce five distinct mutants: Avr4pBLCAT2_907-176, Avr4pBLCAT2_809-176, Avr4pBLCAT2_789-176, Avr4pBLCAT2_429-176 and Avr4pBLCAT2_302-176. The transformants elicited different chloramphenicol acetyl transferase (CAT) activities.

Key words: Avidin, AVR4 promoter, chloramphenicol acetyl transferase.

Recombinant plasmid and vectors

Chloramphenicol acetyltransferase plasmid vector (pBLCAT2) and AVR4 promoter region ligated to pBLCAT2 to produce a recombinant plasmid Avr4pBLCAT2 was obtained from the Dept of Molecular Medicine University of Jyvaskyla, Finland. Stock culture of E. coli HB101 harbouring pBR322 plasmid was obtained from our laboratory.

The nucleotide sequence AVR4 promoter region investigated was obtained from EMBL database search with accession number Z22883 and used to map the cleavage sites of the restriction endonucleases used (Figure 1). 

Figure 1. Avidin related gene 4 (Avr4) promoter region showing the map positions of consensus response elements from transcription start site and restriction enzyme cleavage sites.

Creation of 5' and 3' overhanged Avr4pBLCAT2 plasmids

A 5' overhang of Avr4pBLCAT2 susceptible to Exonuclease III deletion and 3' overhang resistant to the enzyme activity were obtained by SalI and SphI digestion, respectively. The linear plasmids were subjected to Exonuclease III digestion at 25⁰C according to manufacturer's instructions. The assay mixtures were incubated at different time intervals to allow sequential deletion of response elements of Avr4 promoter within each recombinant plasmid in 5' - 3' direction. The incubation period ranged from 29 - 59 min and was terminated by the addition of 2ml 0.5M EDTA at 69⁰C for 10min. This was followed by agarose gel electrophoresis and the desired bands were excised and eluted by using the QIAEX II (R) method (QIAGEN Inc, Canada), and re-ligated. E. coli DH5a cells were made competent prior to transformation with the plasmids according to the protocol of Hanahan et al. (1983).

Each AVR4pBLCAT2 mutant was digested with a pair of restriction enzymes to confirm the deletion of an upstream response element and the presence of the expected terminal response element as indicated in Figure 1. For instance a pair of DsaI and StyI was used to confirm the absence of HSE and the presence API as the terminal response element in AVR4CAT2 mutant 1 (API-API?-APRE-PREIV-HSE-pLCAT2).

Chloramphenicol acetyl transferase (CAT) assay

CAT activity in vitro, as a measure of chloramphenicol degradation in broth was determined by modifying the protocol of Charles and Alan (1999). Each Avr4 E.coli transformant (10⁷ cells) was grown aerobically with shaking at 30⁰C in LB medium containing 20 mg/ml chloramphenicol (Sigma, USA). At 2 hourly intervals cells were pelleted by centrifugation (3000 rpm for 5 min) and aliquots of supernatants were extracted with ethylacetate (equal volume). The resulting organic phase was dried under reduced pressure and heat and the extract reconstituted with 20 ml ethylacetate. The concentration of chloramphenicol in the reconstituted extract was determined by extrapolation from chloramphenicol standard curve as previously described (Shaw, 1975) CAT activity was measured as the micrograms of chloramphenicol degraded per hour under the assay conditions.

In vitro Exonuclease III deletion of the 5' and 3' overhanged Avr4pBLCAT2 generated five distinct mutants at different incubation periods (Table 1). The CAT activities of the constructs are recorded in Figure 2.  

     Table 1. AVR4 promoter constructs generated at different time intervals.

                         Figure 2. Mean CAT activity of the Avr4 promoter deletion constructs.

The present study is aimed at understanding the importance of the upstream response elements in the regulation of avidin gene expression. The obliteration of DsaI cleavage site but the presence of StyI site confirmed API? as the 5’terminal response elements in mutant I.  That mutant 2 has API as its 5’terminal cis element was established by the absence of PstI site within the API? locus of mutant I. Meanwhile, HincII cleavage within the API terminal of mutant 2 was the cause of the 786bp, 1521bp and 2811bp bands observed in this study. Similarly, DdeI produced different fragment length in mutants 3 and 4 to confirm APRE and PREIV as their terminal cis elements.  Finally, the obliteration of AvaII site in mutant 5 confirmed PREIV deletion from the precursor mutant. While StyI established HSE as the terminal regulatory motif in this mutant.  Similar sequential deletion studies have been conducted elsewhere (Ahlroth et al., 2001b).

The observed increased CAT activity among the transformants implies decreased promoter activity with decreased promoter length, indicating the importance of the different promoter elements. 

In conclusion, the successful transformation of E. coli DH5a with all the generated mutants as observed in this study coupled with altered gene expression suggests a possible means of studying avidin gene expression. There is also the need to ascertain the stability of these mutants for long-term storage and use towards elucidating the mechanisms of avidin gene expression as well as applicability in future avidin-biotin technological challenges.