Domain organization and metal ion requirement of the Type IIS restriction endonuclease MnlI

Requirement of divalent cations for DNA cleavage is a general feature of type II restriction enzymes with the exception of few members of this group. A new type II restriction endonuclease has been partially purified from Lactococcus lactis KLDS4. The enzyme was denoted as LlaKI and showed to recognize and cleave the same site as FokI. The enzyme displayed a denatured molecular weight of 50 kDa and behaved as a dimer in solution as evidenced by the size exclusion chromatography. To investigate the role of divalent cations in DNA cleavage by LlaKI, digestion reactions were carried out at different Mg2+, Mn2+, and Ca2+ concentrations. Unlike most of type II restriction endonucleases, LlaKI did not require divalent metal ions to cleave DNA and is one of the few metal-independent restriction endonucleases found in bacteria. The enzyme showed near-maximal levels of activity in 10 mM Tris-HCl pH 7.9, 50 mM NaCl, 10 mM MgCl2, and 1 mM dithiothreitol at 30°C. The presence of DNA modification was also determined and was correlated with the correspondent restriction enzyme.

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Electron microscopic assays of restriction endonuclease cutting, exonuclease digestion, and hybridization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100113317 ◽

1984 ◽

Vol 42 ◽

pp. 686-687

Author(s):

Mark Hannibal ◽

Jacob Varkey ◽

Michael Beer

Keyword(s):

Electron Microscope ◽

Low Temperature ◽

Restriction Endonuclease ◽

Double Helix ◽

Test Material ◽

Electron Microscopic ◽

Exonuclease Iii ◽

Dna Molecule ◽

Linear Molecules ◽

Exonuclease Digestion

Workman and Langmore have recently proposed a procedure for isolating particular chromatin fragments. The method requires restriction endonuclease cutting of the chromatin and a probe, their digestion with two exonucleases which leave complimentary single strand termini and low temperature hybridization of these. We here report simple electron microscopic monitoring of the four reactions involved.Our test material was ϕX-174 RF DNA which is cut once by restriction endonuclease Xho I. The conversion of circles to linear molecules was followed in Kleinschmidt spreads. Plate I shows a circular and a linear DNA molecule. The rate of cutting is shown in Figure 1.After completion of the endonuclease cutting, one portion of the DNA was treated with exonuclease III, an enzyme known to digest the 3' terminals of double helical DNA. Aliquots when examined in the electron microscope reveal a decreasing length of double helix and increasing bushes at the ends.

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Aspects of high-resolution imaging with a scanning ion microprobe

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010014511x ◽

1986 ◽

Vol 44 ◽

pp. 750-751

Author(s):

R. Levi-Setti ◽

J. M. Chabala ◽

Y. L. Wang

Keyword(s):

Metal Ion ◽

Secondary Ion Mass Spectrometry ◽

Chromatic Aberration ◽

Ion Source ◽

Ion Microprobe ◽

Emission Pattern ◽

Intrinsic Resolution ◽

Resolution Imaging ◽

20 Nm ◽

Secondary Ion

We have shown the feasibility of 20 nm lateral resolution in both topographic and elemental imaging using probes of this size from a liquid metal ion source (LMIS) scanning ion microprobe (SIM). This performance, which approaches the intrinsic resolution limits of secondary ion mass spectrometry (SIMS), was attained by limiting the size of the beam defining aperture (5μm) to subtend a semiangle at the source of 0.16 mr. The ensuing probe current, in our chromatic-aberration limited optical system, was 1.6 pA with Ga+ or In+ sources. Although unique applications of such low current probes have been demonstrated,) the stringent alignment requirements which they imposed made their routine use impractical. For instance, the occasional tendency of the LMIS to shift its emission pattern caused severe misalignment problems.

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Scanning ion microscopy images

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010012583x ◽

1987 ◽

Vol 45 ◽

pp. 180-181

Author(s):

R. Levi-Setti ◽

J.M. Chabala ◽

Y.L. Wang

Keyword(s):

Metal Ion ◽

Secondary Emission ◽

Scanning Method ◽

Ion Channeling ◽

Secondary Ions ◽

High Resolution Images ◽

Ion Microscopy ◽

Z Contrast ◽

Focused Beams ◽

Microscopy Images

Finely focused beams extracted from liquid metal ion sources (LMIS) provide a wealth of secondary signals which can be exploited to create high resolution images by the scanning method. The images of scanning ion microscopy (SIM) encompass a variety of contrast mechanisms which we classify into two broad categories: a) Emission contrast and b) Analytical contrast.Emission contrast refers to those mechanisms inherent to the emission of secondaries by solids under ion bombardment. The contrast-carrying signals consist of ion-induced secondary electrons (ISE) and secondary ions (ISI). Both signals exhibit i) topographic emission contrast due to the existence of differential geometric emission and collection effects, ii) crystallographic emission contrast, due to primary ion channeling phenomena and differential oxidation of crystalline surfaces, iii) chemical emission or Z-contrast, related to the dependence of the secondary emission yields on the Z and surface chemical state of the target.

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