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Presents chapter 18 of the book "Handbook of Microbial Biofertilizers." Main reason why it is not possible to establish comparisons between the physiological or ecological behavior of cyanobacterial strains without perfect identification of such strains or species and sharp knowledge of its taxonomic position; Discussion of the taxonomic study of indigenous cyanobacterial strains growing naturally in rice fields located both in Spain and in Argentina; Measurement of nitrogen fixation in the field.

Poly(ADP-ribose) polymerase (PARP-1) plays a crucial role in DNA repair and interacts with many DNA replication/repair factors, including the proliferating cell nuclear antigen (PCNA), a protein involved in many DNA transactions. The association between these proteins in vitro results in the inhibition of PARP-1 activity and of PCNA-dependent DNA synthesis carried out by DNA polymerase δ. In vivo, the interaction of the two proteins has been shown to increase after DNA damage. The activity of PCNA is regulated by p21waf1/cip1, which binds the interdomain connector loop of PCNA in the same region involved in the interaction of PCNA with DNA polymerase δ. This feature enables p21 to compete with DNA polymerase δ to regulate the differential inhibition of DNA replication vs. DNA repair. The finding that PARP- 1 is associated with both PCNA and p21 suggests a possible cooperation of PARP-1 and p21 in regulating the functions of PCNA during DNA replication/repair. [ABSTRACT FROM AUTHOR]

Proliferating cell nuclear antigen (PCNA) is a homotrimeric protein adopting a ring structure that may encircle DNA. In this form, PCNA functions as a sliding platform to which different types of catalytic and regulatory proteins are tethered to perform DNA transactions such as replication and repair synthesis, methylation, chromatin assembly and remodeling, as well as sister chromatid cohesion. In addition, PCNA coordinates DNA metabolism with cell cycle progression by interacting with cyclins, cyclin-dependent kinases (CDK), and CDK inhibitors. PCNA participates in different pathways of DNA repair, including nucleotide and base excision repair, as well as mismatch repair, by interacting with proteins involved in these processes. A fundamental step in DNA repair involves the transition of PCNA from a freely soluble nucleoplasmic form, to a chromatin-bound form associated with repair sites. This chapter describes biochemical and immunofluorescence methods for detection of the chromatin-bound form of PCNA involved in DNA repair. Cellular fractionation and nuclear extraction procedures are provided for Western blot analysis, as well as for protein-protein interaction studies. An in situ extraction protocol is described for immunostaining, fluorescence microscopy and flow cytometric analyses of nuclear localization, and cell cycle distribution of PCNA associated with DNA repair sites. [ABSTRACT FROM AUTHOR]

The cyclin-dependent kinase (CDK) inhibitor p21CDKN1A (also known as p21waf1/cip1) is a well known player of the G1 and G2 phase cell cycle checkpoints, which are activated in response to DNA damage. In addition, p21 interacts directly with proliferating cell nuclear antigen (PCNA), thereby inhibiting DNA replication. More controversial is the role of p21 in DNA repair, since both inhibition of and requirement for nucleotide excision repair have been suggested. Since the DNA repair process occurs at discrete nuclear foci in a chromatin-bound compartment, a suitable extraction procedure is necessary to investigate the association of p21 with PCNA in these structures. This chapter focuses on biochemical and immunofluorescence methods to analyze the recruitment of p21 protein to DNA repair foci. Cellular fractionation and subsequent nuclear extraction procedures are described for Western blot analysis of p21 recruitment, as well as for protein-protein interaction studies. An in situ extraction protocol is also described for immunofluorescence microscopy and flow cytometric analyses of nuclear localization and cell cycle distribution of p21 recruited to DNA repair foci. The combination of these methodologies is extremely powerful to investigate in more detail the role of p21 in the UV-induced DNA damage response. [ABSTRACT FROM AUTHOR]