Giardia lamblia, is a diplomonad, with 2 nuclei and is often referred to as a "deep branching eukaryote" as it diverged out of the main evolutionary tree long before the other eukaryotes. As a result, this oraganism has a number of unique features which have become more "organized" in the higher eukaryotes. One of the most unique features of Giardia is its lack of organellar structures as for example a well defined mitochondria, Golgi bodies and endoplasmic reticulum, in spite of being an eukaryote. Traces of marker proteins from these organelles and an amazingly developed membrane structure adept to carry out these functions are however present here enabling this organism to be classified as a eukaryote .
Though the initials reports of nuclear matrix go as far back as 1960's, the research on S/MARs as potential regulatory elements come from the works of J. Bode in 1988 [40, 41]. Since then, throughout the eukaryotic world, the S/MARs have been found to play a significant role in the organization of chromatin, and gene regulation. Studies on the recently sequenced Giardia genome have shown the genome to be unique in its own way. The protist has 5 chromosomes, and almost 9000 ORFs packed into a small genome of 12 Mb length. It has been seen that the parasite has no homolog for H1 which is the universal linker for compacting chromatin in the nuclei . In this scenario, the study of Scaffold/Matrix attachment region in this parasite can shed adequate light on the chromatin organization in this organism. We did a preliminary screen on the G.lamblia genome with available S/MAR prediction tools. When the common regions predicted by at least 2 tools were taken into consideration, we were able to shortlist at least 15 putative S/MAR regions. To prove this DNA fragments were indeed nuclear matrix dependent fragments we did the PCR based assay, which showed that 10 out of 15 putative S/MARs where actually associated with the nuclear matrix of Giardia. This showed that the false positive rate of our strategy was about 33%. Assuming that the distance between the S/MARs in this genome can range from 50-160 kb, as seen in Table 1, we expect about 110 S/MARs in the entire genome of Giardia. The combination of computational tools correctly predicted only 10% of the total number of the expected S/MARs. This indicates that the S/MAR prediction tools that can be used with accuracy on the higher eukaryotic genome, in most of the instances are not very accurate in predicting lower eukaryotic S/MARs. Experimental methods are an absolute necessity in correctly identifying these elements from the lower eukaryotic genomes. The computational tools for S/MAR predictions can only be used as an initial screen for scanning the genome of the protists for presence or absence of S/MARs, but the actual confirmation is achieved only by experimental methods. Of the 10 S/MARs, 8 also showed positive nuclear matrix binding property in south western blots. Among these, 7 S/MARs which showed positive binding both in PCR as well as south western assay were indeed true S/MARs. It now remains to test these Giardia S/MARs for chromosomal organization studies.
One of the major properties of S/MARs is chromosome organization, anchoring and maintenance of higher order structure . This is achieved by the proteins in the nuclear matrix which bind to the S/MARs thereby allowing it to carry out these functions. The proteins in the nuclear matrix are involved in a host of different functions, including DNA replication and repair . Of these the S/MAR binding proteins are (S/MARBP) are of utmost importance as they regulate transcription, replication, repair and regulation of gene expression . One of the GlSMARs, GlSMAR7 bound to a proteasome subunit 8 as shown by our mass spectrometry results. The 26 S proteasome is an eukaryotic ATP-dependent protease complex of 2000 kd which is reported to be present in the nuclear matrix in mouse myoblasts . As seen in Figure 5, the conserved domain in the 26 S proteasome subunit 8 in Giardia was a AAA domain belonging to the ATPase binding protein superfamily. These proteins perform a diverse range of functions in the cell starting vesicle fusion, peroxidase biogenesis  to DNA repair . Thus it is not unlikely that this protein would be associated with S/MARs and have DNA binding properties. There have been reports on the proteasome 20 S of Giardia lamblia [47, 48], where Emmerlich et al showed the 14 subunits making up this proteasome structure. Though the annotated genome of Giardia shows the presence of several of the proteasome 26 S subunits, no detailed analysis has been done on these proteins in Giardia. A detailed phylogenetic analysis of another AAA ATPase domain containing protein Midasin has been studied by Gallego et al. . This protein is conserved thoughout eukaryotes and plays the role of a nuclear chaperone in most organisms. One of the proteins found to be associated with S/MARs from yeast to humans, is the SAF Box domain containing protein. As reported by Kipp et al in 2000 , SAF-A binds to S/MARs through a novel conserved protein domain. A search in http://www.eupathDB.org for proteins having the SAF box or the SAP domain showed that 47 such proteins were present in the different protozoan genomes (Cryptosporidium, Plasmodium, Toxoplasma, Entamoeba and Trichomonas). Thus it is likely that these genomes will also have S/MAR like elements in their genome. However, when searched in the Giardia genome, this SAF/SAP domain containing protein was not present. Our experimental results discussed in this work indicate that Giardia has S/MAR binding protein (26 S proteasome subunit 8) which does not have a SAF/SAP domain, but has nucleotide binding domains. While it is possible that in a recently sequenced genome, this protein was not annotated, it is also possible that Giardia placed much earlier in the evolutionary scale probably has not yet defined a machinery where these highly conserved domain containing proteins may be present. The presence of S/MARs in Giardia and the absence of SAF box proteins in this organism may also indicate that the early divergence of Giardia during evolution probably.resulted in "missing out" this very conserved protein involved in nuclear architecture.
S/MARs have been found to be associated with not only chromatin anchoring but also with other regions of the genome as introns  and can play a significant role in the regulation of gene expression [52, 53]. Studies on S/MAR in Arabidopsis and maize [54, 55] have shown that the plant genome is not packaged by random gathering into domains of indiscriminate length, but rather, the genome is gathered into specific domains, and a gene consistently occupies a discrete physical section of the genome. The average loop size in Arabidopsis and maize has been estimated as 25 and 45 kb, respectively , though other studies  have suggested smaller domain sizes. Some loops may remain permanently condensed and inactive, even within the euchromatic portions of the genome, whereas others can be extended to produce a transcriptionally poised conformation in appropriately differentiated cells . Our analysis for a genome-wide distribution of S/MARs using different tools indicates that the loop size ranges from 50-160 kbp in Giardia (Table 1). Data on the location of transcribed elements within structural loops at the supragenic level suggest that attachment to the matrix and transcription is not systematically associated [57, 58], though S/MARs are associated with the ends of some DNaseI-sensitive (transcriptionally poised) domains . S/MARs have also been identified within introns of genes [60, 61]. Cockerill et al.  suggested that S/MARs flanking enhancer sequences may act as positive and/or negative regulators of enhancer function. It is presumed that additional specific S/MARs have been further demonstrated in a variety of functional tests to act as insulators , according to the loop domain model, by protecting a loop from the effects of the neighboring chromatin or associated enhancer sequences. Distribution of Giardia S/MARs among the transcription factors also hints at this possibility (data not shown). A much more in-depth study of the S/MARs in lower eukaryotes is required to understand the chromatin dynamics and packing in these organisms.
An observation was made in the study by Linnemann et al in 2009, where it was seen that the S/MARs when present in the 5' region of a gene resulted in a transcript presence, where as those present within the ORF associated with silenced genes. A number of S/MARs in Giardia were also found within the ORFs. The significance of this is not clear. In Entamoeba, such S/MARs were found to have reduced binding ability to nuclear matrix compared to the ones that were present outside ORF (our unpublished data). It is possible that in these early eukaryotes, the genome organization machinery is also in early stages of evolution and the S/MARs within the ORFs are actually the ones which in course of evolution would lose their ability to bind to the nuclear matrix completely.