Although ingenious genetic experiments can reveal much about biological systems, ultimately the details must be studied biochemically. Therefore it was of great interest to isolate the Int and Xis proteins. Such an isolation requires an assay for the proteins. The most reliable assay for Int protein activity is to seek a protein that carries out the entire integration reaction in vitro. As mentioned above, one likely requirement for such a reaction to proceed in vitro is a high concentration of attB and attP regions. The easiest way to obtain these is to place both on the same DNA molecule.

Can attB and attP be put on the same molecule, just as attL and attR could be put on λatt²? Nash constructed such phage by selecting the product of a rare recombination event in a region of nonhomology between a λdgal-bio containing attB and a λ (Fig.1). Similar to the excision-type of reaction that occurs on λatt2, on λattB-attP an integration-type of reaction can occur. This removes the bio region and leaves the phage considerably more resistant to Mg⁺⁺ chelation or heat. Hence, the parental phage and derivatives that have undergone an integration reaction and have become smaller can easily be distinguished.

Fig1. The illegitimate recombination by which the λattB-attP phage was generated.

Initial tests with the λattB-attP phage showed that an in vitro integration reaction catalyzed by an extract from cells would work. The experiment was performed by incubating the λattB-attP DNA with a cell extract prepared from heat-pulsed lambda lysogens. Then the DNA was extracted from the mix and used to transfect cells that had been made capable of taking up naked lambda DNA. The phage from the transfected spheroplasts were spread on a lawn of sensitive cells on a plate containing pyrophosphate. Under these conditions, only products from the integrative reaction would produce plaques. This provided an extremely sensitive assay for the integration reaction. Later, as the assay conditions were improved, the integration reaction could be assayed merely by physical quantitation of the DNA products. The locations of restriction enzyme cleavage sites in the DNA are rearranged by the integration reaction. This creates new sizes of restriction fragments that can be detected by separating the resulting fragments by electrophoresis (Fig.2).

Fig2. Digestion with a restriction enzyme generates different fragments from the λattB-attP phage and its integration "product," lambda and the minicircle.

The in vitro integration reaction required Mg⁺⁺, ATP, and spermidine. More careful examination showed that the use of supercoiled DNA eliminated the requirement for ATP and Mg⁺⁺. The assays permitted the purification and characterization of biologically active Int protein, and analogous experiments have been done with the λatt2 for the purification of Xis protein.

اخر الاخبار

اخبار العتبة العباسية المقدسة

العتبة العباسية المقدسة تنشر لافتات الحزن والعزاء بذكرى شهادة الإمام الحسن المجتبى (عليه السلام)

كلية التمريض في جامعة العميد تحصل على الاعتماد البرامجي الكامل من وزارة التعليم

العتبة العباسية المقدسة تعلن تهيئة مساحات جديدة قرب الحرم الطاهر استعدادًا لزيارة الأربعين

ملاكات العتبة العباسة المقدسة تواصل أعمال تهيئة صحن أمّ البنين (عليها السلام) استعدادًا لزيارة الأربعين

المزيد

الآخبار الصحية

ماذا يحدث لضغط الدم مع تناول بذور الشيا؟

الأولى من نوعها.. حقنة ثورية تنهي معاناة فقدان السمع نهائيا !

7 علامات تدل على نقص معدن مهم في الجسم.. تعرف عليها

الجسم يستعيد الوزن الذي فقده بعد أسابيع من وقف أدوية التنحيف

المزيد

الآخبار التكنلوجية

أمواج تسونامي تصل إلى هاواي بعد زلزال كامتشاتكا !

تشات جي بي تي يتجاوز اختبار "أنا لست روبوتا" المرعب ؟!

أستراليا تعتزم حظر استخدام "يوتيوب" لهذه الفئة العمرية

دراسة تكشف تأثير استخدام الهواتف بسن مبكرة على الصحة النفسية

المزيد

مواضيع ذات صلة

Structure of the Intasome

Inhibition By a Downstream Element

Demonstrating Xis is Unstable

The Protein Kinase Domain

Posttranslational Modifications

The Double att Phage, att2

Transducing Phage Carrying Genes Other than gal and bio

Incorrect Excision and gal and bio Transducing Phage

Isolation of Excision-Deficient Mutants

Amplification Techniques in Molecular Biology

Characteristics of Nucleic Acids

solation of Integration-Defective Mutants