Tissue stem cells and cancer stem cells: potential implications for gastric cancer.

Gastric cancer remains the second leading cause of death in the world today, making the search for its molecular and cellular basis an important priority. Though recognition of the tight link between inflammation and tumorigenesis is centuries old, only recently are the pieces of the etiological puzzle beginning to fall together. Recent advances in gastric stem cell biology appear to be central to this slowly resolving puzzle. At least two types of stem cells may be important. Resident adult or tissue stem cells may, in a chronically inflamed environment, slowly acquire a series of genetic and epigenetic changes that lead to their emergence as ''cancer stem cells''. This scenario has not yet been proven experimentally, although the first step, prospective recognition of a gastric stem cell has recently been conquered. Alternatively, the setting of chronic inflammatory stress and injury may lead to loss of the indigenous gastric stem cells from their niches; bone marrow derived stem cells may then be recruited to and engraft into the gastric epithelium. Such recruited cells have the potential to contribute to the tumor mass. Indeed, evidence supporting this scenario has been published. Here, we review these recent findings and discuss implications for the future.

Peroxisome proliferator activated receptor gamma in colonic epithelial cells protects against experimental inflammatory bowel disease.

INTRODUCTION: Peroxisome proliferator activated receptor gamma (PPARgamma) is expressed in epithelial cells, macrophage, and T and B lymphocytes. Ligand induced activation of PPARgamma was reported to attenuate colitis activity but it is not clear whether this protection is mediated by epithelial or leucocyte PPARgamma. METHODS: Mice with targeted disruption of the PPARgamma gene in intestinal epithelial cells, generated using a villin-Cre transgene and floxed PPARgamma allele and designated PPARgamma(DeltaIEpC), were compared with littermate mice having only the PPARgamma floxed allele with no Cre transgene that expressed PPARgamma in the gut, designated PPARgamma(F/F). Colitis was induced by administering dextran sodium sulphate (DSS) and the two mouse lines compared for typical symptoms of disease and expression of inflammatory cytokines. RESULTS: PPARgamma(DeltaIEpC) mice displayed reduced expression of the PPARgamma target genes ADRP and FABP in the gut but were otherwise normal. Increased susceptibility to DSS induced colitis, as defined by body weight loss, colon length, diarrhoea, bleeding score, and altered histology, was found in PPARgamma(DeltaIEpC) mice in comparison with PPARgamma(F/F) mice. Interleukin (IL)-6, IL-1beta, and tumour necrosis factor alpha mRNA levels in colons of PPARgamma(DeltaIEpC) mice treated with DSS were higher than in similarly treated PPARgamma(F/F) mice. The PPARgamma ligand rosiglitazone decreased the severity of DSS induced colitis and suppressed cytokine production in both PPARgamma(F/F) and PPARgamma(DeltaIEpC) mice. CONCLUSIONS: These studies reveal that PPARgamma expressed in the colonic epithelium has an endogenous role in protection against DSS induced colitis and that rosiglitazone may act through a PPARgamma independent pathway to suppress inflammation.

Fire and ICE: the role of pyrin domain-containing proteins in inflammation and apoptosis.

The genetic bases for several human autoinflammatory syndromes have recently been identified, and the mutated proteins responsible for these diseases are rapidly being characterized. Here, we examine two of these newly identified proteins, pyrin (also called marenostrin, product of the familial Mediterranean fever locus, MEFV) and cryopyrin (product of the CAIS1 locus, and mutated in familial cold urticaria, Muckle Wells syndrome and chronic infantile neurological cutaneous and articular syndrome). Both pyrin and cryopyrin contain an N-terminal domain that encodes a death domain-related structure, now known as the pyrin domain, or PyD. We trace the molecular interactions mediated by these PyDs, examine the evolution of the family of molecules containing this domain, and discuss the function of PyD-containing proteins and their homologues. Synthesis of the available data indicates that both pyrin and cryopyrin interact via their PyDs with a common adaptor protein, ASC. ASC itself participates in at least three important cellular processes: apoptosis, recruitment and activation of pro-caspase-1 (with associated processing and secretion of IL-1beta), and activation of NF-kappaB (a transcription factor involved in both initiation and resolution of the inflammatory response). Through PyD:PyD interactions, pyrin and cryopyrin, as well as several related, but still uncharacterized PyD containing proteins, appear to modulate the activity of all three of these processes, each of which plays a crucial role in the inflammatory pathways that characterize the innate immune system.

Interaction between pyrin and the apoptotic speck protein (ASC) modulates ASC-induced apoptosis.

Patients with familial Mediterranean fever suffer sporadic inflammatory attacks characterized by fever and intense pain (in joints, abdomen, or chest). Pyrin, the product of the MEFV locus, is a cytosolic protein whose function is unknown. Using pyrin as a "bait" to probe a yeast two-hybrid library made from neutrophil cDNA, we isolated apoptotic speck protein containing a caspase recruitment domain (CARD) (ASC), a proapoptotic protein that induces the formation of large cytosolic "specks" in transfected cells. We found that when HeLa cells are transfected with ASC, specks are formed. After co-transfection of cells with ASC plus wild type pyrin, an increase in speck-positive cells is found, and speck-positive cells show increased survival. Immunofluorescence studies show that pyrin co-localizes with ASC in specks. Speck localization requires exon 1 of pyrin, but exon 1 alone of pyrin does not result in an increase in the number of specks. Exon 1 of pyrin and exon 1 of ASC show 42% sequence similarity and resemble death domain-related structures in modeling studies. These findings link pyrin to apoptosis pathways and suggest that the modulation of cell survival may be a component of the pathophysiology of familial Mediterranean fever.

cDNA cloning, chromosomal localization and evolutionary analysis of mouse vacuolar ATPase subunit D, Atp6m.

The multi-subunit vacuolar ATPase pump uses ATP hydrolysis to move protons into membrane bound compartments. The pump is involved in a variety of cellular functions, including regulation of cytosolic pH, vesicular transport, endocytosis, secretion, and apoptosis. Here, we describe the cDNA cloning and chromosomal mapping of subunit D of murine V-ATPase. The mouse gene, designated Atp6m, maps to Chromosome 12, in a region of high homology with human chromosome 14q24. Evolutionary analysis of subunit D orthologs in a variety of other species reveals that this is a highly conserved protein that has been under remarkably strong negative selection during evolution, most likely reflecting its critical role in multiple cellular processes.

Humans and old world monkeys have similar patterns of fetal globin expression.

The expression of epsilon- and gamma-globin mRNA and protein has been determined in three Old World monkey species (Macaca mulatta, Macaca nemestrina, and Cercopithecus aethiops). Using RT-PCR with primers for epsilon- and gamma-globin, both mRNAs were detected in early fetal stages, whereas at 128 days (85% of full term), only gamma was expressed. High-performance liquid chromatography was used for separation and quantitation, and matrix-assisted laser desorption/ionization mass spectrometry was used for identification of globin polypeptides. An alpha-globin polymorphism was observed in all of the species examined. During fetal life, gamma-globin was the predominant expressed beta-type globin. The red blood cells of infants still contained substantial amounts of gamma-globin, which declined to negligible levels in 14 weeks as beta-globin expression reached adult values. The ratio of gamma1- to gamma2-globins (equivalent to Ggamma/Agamma in humans) was approximately 2.5, similar to the Ggamma/Agamma ratio observed in humans. Thus, gamma-globin gene expression in these Old World monkeys species has three features in common with human expression: expression of both duplicated gamma genes, the relative preponderance of gamma1 over gamma2 expression, and the delay of the switch from gamma- to beta-globin until the perinatal period. Thus, the catarrhines seem to share a common pattern of developmental switching in the beta-globin gene cluster, which is distinct from the timing of expression in either prosimians or the New World monkeys. Our results indicate that an Old World monkey, such as Rhesus, could serve as a model organism (resembling humans) for experimentally investigating globin gene expression patterns during the embryonic, fetal, and postnatal stages.

Model for the fetal recruitment of simian gamma-globin genes based on findings from two New World monkeys Cebus apella and Callithrix jacchus (Platyrrhini, Primates).

The originally embryonic gamma-globin locus duplicated and acquired a novel (fetal) pattern of expression in a defined time period (55-40 million years ago) during primate phylogeny. The objective of this study was to determine some of the factors that led to first the emergence of fetal gamma specificity and then the maintenance of different fetal gamma expression patterns in extant simian primates (e.g., human, capuchin monkey). Analyses focused on two platyrrhine (New World monkey) species: the common marmoset (Callithrix jacchus) and the brown capuchin monkey (Cebus apella), each of which has paired, non-allelic gamma loci (5'-gamma 1-gamma 2-3'). Quantitation of beta-type globin mRNAs expressed in a 4.5 week old embryo of Callithrix jacchus revealed that in addition to its primary epsilon-globin message, considerable amounts of gamma 1 message and just trace levels of gamma 2 message are present. In contrast, analyses of gamma-globin messenger RNAs expressed in a Cebus apella fetal liver indicated that gamma 2 expression is at least 120 times greater than gamma 1 expression. Using a luciferase reporter and a transient assay system, the strengths of gamma 1 and gamma 2 promoter fragments of Cebus apella were compared in erythroid (K562) and non-erythroid (HeLa) cell lines. Due to the lack of chromatin repression in a transient expression system, the results do not fully recapitulate globin expression. However, the results suggest that sequences contained within the Cebus gamma 1 and gamma 2 proximal promoter regions (-200 to +1 bp) can direct gamma transcription in both cell lines. In K562 and, to a lesser extent, in HeLa cells Cebus gamma 2 promoter fragments were significantly stronger (P < 0.01) than gamma 1 promoter fragments. This is consistent with the fact that the Cebus gamma 1 promoter contains several mutations, including a proximal CCAAT box mutation (CCAAT-->CCAAc). The epsilon-gamma 1 intergenic distances in these platyrrhines (5.4 kb in Cebus apella and 6.9 kb in Callithrix jacchus) are short, supporting the inference that it was also short in the stem simian primates. The results suggest that immediately following the gamma duplication, the gamma 1 gene of the stem simians was still embryonic and the downstream gamma 2 gene was largely silent. A further inference is that once gamma 2 accumulated regulatory mutations that disrupted binding of fetal repressors, gamma 2 was expressed fetally and, through gene conversion, passed these characteristics to the gamma 1 gene. The fetal expression of gamma 1 is most evident in catarrhines (Old World monkeys and hominoids), which preferentially express the gamma 1 locus during fetal life.

Analysis of linked human epsilon and gamma transgenes: effect of locus control region hypersensitive sites 2 and 3 or a distal YY1 mutation on stage-specific expression patterns.

Stage-specific expression of the human beta-like globin genes is controlled by interactions between regulatory elements near the individual genes and additional elements located upstream in the Locus Control Region (LCR). Elucidation of the mechanisms that govern these interactions could suggest strategies to reactivate fetal (gamma) or embryonic (epsilon) genes in individuals with severe hemoglobinopathies. This study extends an earlier analysis of a transgenic construct, HS3epsilon gamma, testing: (A) the effect of substitution of HS2 for HS3 on stage-specific expression of the epsilon and gamma genes and, (B) the role of an evolutionarily conserved YY1 binding site in transcriptional regulation of the gamma gene. The data show that both HS3epsilon gamma and HS2epsilon gamma can individually support embryonic expression of epsilon and fetal expression of Agamma. Thus, the cis regulators of distinct expression patterns for epsilon and gamma are likely to reside near the genes, rather than in specific hypersensitive sites of the LCR. Alterations in Agamma expression patterns observed in transgenic lines carrying a construct with a mutation in a conserved YY1 binding site at -1086 indicate that this site might function to facilitate active transcription of the gamma gene in fetal life.

Targeted disruption of the mouse villin gene does not impair the morphogenesis of microvilli.

The small intestine is functionally dependent on the presence of the brush border, a tightly packed array of microvilli that forms the amplified apical surface of absorptive cells. In the core of each microvillus, actin filaments are bundled by two proteins, villin and fimbrin. Previous in vitro studies using antisense approaches indicated that villin plays an important role in the morphogenesis of microvilli. To examine the in vivo consequences of villin deficiency, we disrupted the mouse villin gene by targeted recombination in mouse embryonic stem cells. A beta-galactosidase cDNA was also introduced into the villin locus by the targeting event. Homozygous villin-deficient mice are viable, fertile, and display no gross abnormalities. Intact microvilli are present in the small intestine, colon, kidney proximal tubules, and liver bile canaliculi. Although subtle ultrastructural abnormalities can be detected in the actin cores of small intestinal microvilli, localization of sucrase isomaltase, brush border myosin I, and zonula occludens I to the microvillar surface of the small intestine is normal. Thus, in vivo, villin plays a minor or redundant role in the generation of microvilli in multiple absorptive tissues.

Familial Mediterranean fever and hyperimmunoglobulinemia D syndrome: two diseases with distinct clinical, serologic, and genetic features.

OBJECTIVE: To determine whether the 2 periodic febrile syndromes familial Mediterranean fever (FMF) and hyperimmunoglobulinemia D syndrome (HIDS) are distinct diseases. METHODS: Clinical manifestations of the diseases were analyzed by physicians experienced with FMF and HIDS. Serum immunoglobulin (Ig) levels were studied in 70 patients with FMF using nephelometry or ELISA and compared with Ig levels in 50 patients with HIDS. Genetic linkage of HIDS with the chromosome 16 polymorphic locus RT70, currently used for refined localization of the FMF susceptibility gene (MEFV), was studied in 9 HIDS families (18 patients) using polymerase chain reaction amplification and gel electrophoresis. RESULTS: The main clinical features distinguishing FMF from HIDS were lymphadenectomy, skin eruption, and symmetrical oligoarthritis in HIDS, and monoarthritis, peritonitis, and pleuritis in FMF. Increased IgG levels were found in 12 patients with FMF (17%), IgA in 16 (23%), IgM in 9 (13%), and IgD in 9 (13%), significantly lower than the prevalence reported for HIDS. We found no evidence for genetic linkage between HIDS and the chromosome 16 marker RT70. CONCLUSION: HIDS and FMF are different entities, clinically, immunologically, and genetically.

Construction of a 1-Mb restriction-mapped cosmid contig containing the candidate region for the familial Mediterranean fever locus (MEFV) on chromosome 16p 13.3.

In this paper we describe the assembly and restriction map of a 1.05-Mb cosmid contig spanning the candidate region for familial Mediterranean fever (FMF), a recessively inherited disorder of inflammation localized to 16p13.3. Using a combination of cosmid walking and screening for P1, PAC, BAC, and YAC clones, we have generated a contig of genomic clones spanning approximately 1050 kb that contains the FMF critical region. The map consists of 179 cosmid, 15 P1, 10 PAC, 3 BAC, and 17 YAC clones, anchored by 27 STS markers. Eight additional STSs have been developed from the approximately 700 kb immediately centromeric to this genomic region. Five of the 35 STSs are microsatellites that have not been previously reported. NotI and EcoRI mapping of the overlapping cosmids, hybridization of restriction fragments from cosmids to one another, and STS analyses have been used to validate the assembly of the contig. Our contig totally subsumes the 250-kb interval recently reported, by founder haplotype analysis, to contain the FMF gene. Thus, our high-resolution clone map provides an ideal resource for transcriptional mapping toward the eventual identification of this disease gene.

Evolution of a fetal expression pattern via cis changes near the gamma globin gene.

One basis for the evolution of organisms is the acquisition of new temporal and spatial domains of gene expression. Such novel expression domains could be generated either by cis sequence changes that alter the complement of trans-acting regulators binding to control elements or by changes in the expression patterns of one or more of the regulatory (trans) factors themselves. The gamma globin gene is a prime example of a gene that has undergone a distinct change in temporal expression at a defined time in evolution. Approximately 35-55 million years ago, the previously embryonic gamma gene acquired a fetal expression pattern. This change occurred in a simian primate ancestor after the separation of simian and prosimian primates but before the further separation of the major simian lineages; thus, the (prosimian) galago gamma gene retains the ancestral embryonic expression pattern, whereas the (simian) human gamma gene is fetal. This analysis of galago and human gamma genes in transgenic mice demonstrates that cis changes in sequences within a 4.0-kilobase region surrounding the gamma gene were responsible for the evolution of a novel fetal expression pattern in the gamma globin genes of simian primates.

Phylogenetic footprinting of hypersensitive site 3 of the beta-globin locus control region.

Hypersensitive site 3 (HS3) of the beta-like globin locus control region has been implicated as an important regulator of the beta-like globin genes, but the trans factors that bind HS3 have only been partially characterized. Using a five-species alignment (human, galago, rabbit, goat, and mouse) that represents 370 million years of evolution, we have identified 24 phylogenetic footprints in the HS3 core and surrounding regions. Probes corresponding to the human sequence at each footprint have been used in binding studies to identify the nuclear factors that bind within and near these conserved sequence elements. Among the high-affinity interactions observed were several binding sites for proteins with repressor activity, including YY1, CCAAT displacement protein, and G1/G2 complexes (uncharacterized putative repressors) and several binding sites for the stage selector protein. To complement this analysis, orthologous galago sequences were also used to derive probes and the pattern of proteins binding to human and galago probes was compared. Binding interactions differing between these two species could be responsible for the different expression patterns shown by the two gamma genes (galago gamma is embryonic; human gamma is fetal). Alternatively, binding interactions that are conserved in the two species may be important in the regulation of common expression patterns (eg, repression of gamma in adult life).

The complete sequences of the galago and rabbit beta-globin locus control regions: extended sequence and functional conservation outside the cores of DNase hypersensitive sites.

The locus control region (LCR) of mammalian beta-globin genes covers at least 17 kb at the 5' end of the gene cluster and has been implicated in chromatin domain opening, enhancement, and insulation from neighboring sequences. Functional dissection of the LCR has defined the minimal cores for four of the five major DNase hypersensitive sites (HSs) that mark this regulatory region. To examine fully the patterns of conserved sequences in the mammalian homologs to the beta-globin LCR, we determined the complete DNA sequence of the galago beta-globin LCR and completed previously unsequenced regions of the rabbit LCR. Simultaneous alignment of these sequences with the human, goat, and mouse LCRs revealed conserved sequences (phylogenetic footprints) detected using three largely independent methods. The most highly conserved segments are found both within the HS cores and in some but not all regions flanking the cores. These results argue for an extended pattern of well-conserved sequences, many of which lie outside the minimal cores, and we show that a key sequence required for domain opening by the region including HS3 maps about 1 kb 5' to the minimal core. Differential phylogenetic footprints, containing sequences conserved in nonhuman mammals but not in humans, are found primarily around HS3, consistent with some species-specific differences in function that may be important for differences in hemoglobin switching during development.

Dynamics of regulatory evolution in primate beta-globin gene clusters: cis-mediated acquisition of simian gamma fetal expression patterns.

Phylogenetic reconstructions by parsimony were carried out on an enlarged body of primate gamma1 and gamma2-globin sequences. The results confirm that gamma1 and gamma2 arose from a tandem duplication in an ancient simian lineage ancestral to both platyrrhines (New World monkeys) and catarrhines (Old World monkeys and hominoids). Gene conversions between the two gamma homologs were frequent over the gamma gene proper but less frequent over the 5' flanking and very infrequent over the 3' flanking regions. The ancient platyrrhine conversion in the most distal 5' flanking region had the polarity of gamma2-->gamma1. Recent platyrrhine conversions between 5' regulatory sequences were very infrequent, in striking contrast to catarrhines which have large, uninterrupted stretches of converted 5' regulatory sequences. Comparisons of reconstructed ancestral primate and simian gamma promoter sequences revealed an accumulation of 21 nucleotide substitutions concentrated in or near cis-elements that may have mediated the change from embryonic to fetal gamma expression. Almost all 21 substitutions were retained in the lineages leading to functional gamma genes of extant catarrhines (both gamma1 and gamma2) and platyrrhines (most often gamma2). Fewer of these simian specific substitutions were retained in the platyrrhine gamma1 genes and new mutations occurred more often in the platyrrhine gamma1 than gamma2 promoters.

Locus control regions of mammalian beta-globin gene clusters: combining phylogenetic analyses and experimental results to gain functional insights.

Locus control regions (LCRs) are cis-acting DNA segments needed for activation of an entire locus or gene cluster. They are operationally defined as DNA sequences needed to achieve a high level of gene expression regardless of the position of integration in transgenic mice or stably transfected cells. This review brings together the large amount of DNA sequence data from the beta-globin LCR with the vast amount of functional data obtained through the use of biochemical, cellular and transgenic experimental systems. Alignment of orthologous LCR sequences from five mammalian species locates numerous conserved regions, including previously identified cis-acting elements within the cores of nuclease hypersensitive sites (HSs) as well as conserved regions located between the HS cores. The distribution of these conserved sequences, combined with the effects of LCR fragments utilized in expression studies, shows that important sites are more widely distributed in the LCR than previously anticipated, especially in and around HS2 and HS3. We propose that the HS cores plus HS flanking DNAs comprise a 'unit' to which proteins bind and form an optimally functional structure. Multiple HS units (at least three: HS2, HS3 and HS4 cores plus flanking DNAs) together establish a chromatin structure that allows the proper developmental regulation of genes within the cluster.

Fetal globin expression in New World monkeys.

Reverse phase chromatography of the globin chains of adult, newborn, and fetal erythrocytes from three species of New World monkeys (Cebus apella, Aotus azarae, and Callithrix jacchus) representing three of the seven platyrrhine clades showed that gamma-globin expression was fetal in these animals. The globins were identified by a combination of chemical sequencing and mass spectrometric analysis. Since gamma-globin expression is fetal in the other major simian branch, the catarrhines, but embryonic in prosimian primates and nonprimate placental mammals, the evolution of fetal recruitment can now be assigned to the period between the simian-prosimian divergence (55 million years ago) and the platyrrhine-catarrhine divergence (35 million years ago). The gamma-globin gene underwent tandem duplication during the same evolutionary epoch, in accord with a model that suggests that the downstream duplicated gamma-gene (gamma2) was free to acquire the mutations necessary for fetal recruitment. Mass spectrometric analysis of tryptic digests of the gamma-globins verified the amino acid sequences deduced from genomic sequencing. Detailed analysis of high performance liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry data showed that gamma2-globin in Cebus was expressed to a far greater extent than gamma1-globin, supporting inferences drawn from a study of the promoter sequences. A "pre-gamma"-globin was observed in C. apella and shown to be primarily the glutathionyl adduct. The other species, A. azarae and C. jacchus, also express only one gamma-globin polypeptide. This work provides biochemical evidence of an evolutionary trend in the platyrrhines to alter the duplicated gamma-globin gene locus so that only one gamma-globin polypeptide is expressed.

Reduction of two functional gamma-globin genes to one: an evolutionary trend in New World monkeys (infraorder Platyrrhini).

Nucleotide sequences were determined for the gamma1- and gamma2-globin loci from representatives of the seven anciently separated clades in the three extant platyrrhine families (Atelidae, Pitheciidae, and Cebidae). These sequences revealed an evolutionary trend in New World monkeys either to inactivate the gamma1 gene or to fuse it with the gamma2 gene, i.e. to have only one functional fetally expressed gamma gene. This trend is clearly evident in six of the seven clades: (i) it occurred in atelids by deletion of most of the gamma1 gene in the basal ancestor of this clade; (ii-iv) in pitheciid titi, saki, and cebid capuchin monkeys by potentially debilitating nucleotide substitutions in the proximal CCAAT box of the gamma1 promoters and (v and vi) in cebid owl and squirrel monkeys by crossovers that fused 5' sequence from gamma1 with 3' sequence from gamma2. In the five clades with gamma1 and gamma2 loci separated by intergenic sequences (the fifth clade being the cebid marmosets), the gamma2 genes retained an unaltered proximal CCAAT motif and their gamma2 promoters accumulated fewer nucleotide substitutions than did the gamma1 promoters. Thus, phylogenetic considerations indicate that the stem platyrrhines, ancestral to all New World monkeys, had gamma2 as the primary fetally expressed gamma gene. A further inference is that when the earlier stem anthropoid gamma gene duplicated, gamma2 (at its greater downstream distance from epsilon) could evade embryonic activation by the locus control region but could be fetally activated once released by regulatory mutations from fetal repressors.

Evolutionary strategies for the elucidation of cis and trans factors that regulate the developmental switching programs of the beta-like globin genes.

We describe three strategies for the identification of specific cis and trans factors that regulate globin gene expression, all three of which are based on the evolution of the globin genes and their expression patterns. The first approach, phylogenetic footprinting, relies on a search for sequence similarities and is designed to elucidate the factors that control those expression patterns which are shared by orthologous globin genes of all eutherian mammals (e.g., the expression of the epsilon globin genes in the embryonic yolk sac and its repression in fetal and adult hematopoietic tissues). The second approach, differential phylogenetic footprinting, relies on a search for sequence differences. This approach may be of value in identifying the mechanisms underlying the generation of novel expression patterns in specific lineages (e.g., the expression of gamma as a fetal gene in the simian primates in contrast with the embryonic expression of gamma in all other mammals). Finally, motif-based phylogenetic analysis takes into consideration the fact that many transcription factors are quite flexible in the recognition of their cognate sites. The approach allows the detection of functionally conserved binding sites despite their sequence variation.