After the isolation of lac repressor and the demonstration that it bound to DNA, interest turned to the structure of operator. The first questions concerned its size and sequence. Answering such questions is now straightforward with the application of genetic engineering techniques. The questions about the lac operator, however, came to the fore before the era of genetic engineering. Because ingenious techniques were developed and the work directly led to the development of many of the genetic engineering techniques now used, we will review the techniques, now outdated, used to isolate and sequence the lac operator.

Before developing their chemical sequencing method, Gilbert and Maxam sequenced the operator, primarily by direct DNA sequencing methods, whereas somewhat later Reznikoff, Barnes, Abelson, and co-workers sequenced the entire lac regulatory region by purifying and sequencing RNA copies of it (Fig. 1). Gilbert’s method required isolation of pure operator itself. DNA was isolated from phage carrying the lac genes. This DNA was sonicated to fragments about 1,000 base pairs long, repressor was added, and the mixture was passed through a cellulose nitrate filter. The DNA fragments containing operator were bound by repressor and held on the filter whereas the rest passed through. Then the operator-containing fragments were specifically released by adding IPTG to the rinse buffer. Finally, repressor was again bound to these fragments, and the portions of the fragments not protected by repressor were digested by DNAse. The operator was found to be about 20 base pairs long, and it had been purified 2000-fold from the phage or 20,000-fold from the E. coli DNA!

Fig1. The DNA sequence of the lac regulatory region and the binding sites of the proteins that bind there.

The lac operator possesses a symmetrical sequence. This suggested that two of the repressor subunits symmetrically made contact with it (Fig. 2). To probe the structure of lac-operator-repressor complexes, Gilbert and Maxam turned to DNA protection studies. They bound repressor to operator and investigated the reaction of dimethylsulfate with bases of the operator in the complex. Indeed, a symmetrical protection pattern was obtained, but, more important, they noticed that the method could easily and rapidly be adapted to yield the actual sequence of a DNA fragment. From this beginning they developed the chemical method for sequencing DNA.

Fig2. The symmetry in lac operator and how a protein consisting of two identical subunits could bind to it.

اخر الاخبار

اخبار العتبة العباسية المقدسة

قسم الشؤون الدينية يُطلق دورة فقهية دينية لملاكات مستشفى الكفيل

شعبة فاطمة بنت أسد للدراسات القرآنية تعلن نتائج مسابقة (ميثاق الرسول)

شعبة الخطابة الحسينية النسوية تنظّم مسابقة فرقية لطالبات برنامج ثمار العطاء

قسم العلاقات ينظم ورشة عن فن الإقناع والتواصل الفعّال للطلبة المنسقين

المزيد

الآخبار الصحية

تغيير بسيط في نمط التفكير قد يحدث فرقا كبيرا لصحة الدماغ ؟

لماذا ينعشنا المشروب الساخن في الطقس الحار؟

علماء يكتشفون فئة دم فريدة من نوعها في العالم !

لماذا ينصح الأطباء بزيت الزيتون يوميا؟

المزيد

الآخبار التكنلوجية

حساس الإطارات.. ما هو وعلامات تلفه وكيفية صيانته؟

ما معنى أودي Quattro؟ سر الأداء والتميز الهندسي ؟!

فصل الشاحن أم الهاتف أولا؟.. عادات بسيطة تطيل عمر البطارية

تعود للقرن الرابع قبل الميلاد.. اكتشاف بقايا "إيمت" المصرية

المزيد

مواضيع ذات صلة

Targeting of Mitochondrial Proteins

Intra-Golgi Transport and Protein Processing

Cotranslational Protein Translocation Into the Endoplasmic Reticulum

The Migration Retardation Assay and DNA Looping

Repressor Binds to DNA: The Operator is DNA

Detection and Purification of lac Repressor

The Difficulty of Detecting Wild-Type lac Repressor

An Assay for lac Repressor

Proving lac Repressor is a Protein

The Role of Inducer Analogs in the Study of the lac Operon

Background of the lac Operon

Mutagenesis with Chemically Synthesized DNA