Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow

The adhesive mechanisms allowing hematopoietic progenitor cells (HPC) homing to the bone marrow (BM) after BM transplantation are poorly understood. We investigated the role of endothelial selectins and vascular cell adhesion molecule-1 (VCAM-1) in this process. Lethally irradiated recipient mice deficient in both P-and E-selectins (P/E−/−), reconstituted with minimal numbers (≤5 × 104) of wild-type BM cells, poorly survived the procedure compared with wild-type recipients. Excess mortality in P/E−/− mice, after a lethal dose of irradiation, was likely caused by a defect of HPC homing. Indeed, we observed that the recruitment of HPC to the BM was reduced in P/E−/− animals, either splenectomized or spleen-intact. Homing into the BM of P/E−/− recipient mice was further compromised when a function-blocking VCAM-1 antibody was administered. Circulating HPC, 14 hr after transplantation, were greatly increased in P/E−/− mice treated with anti-VCAM-1 compared with P/E−/− mice treated with just IgG or wild-type mice treated with either anti-VCAM-1 or IgG. Our results indicate that endothelial selectins play an important role in HPC homing to the BM. Optimal recruitment of HPC after lethal doses of irradiation requires the combined action of both selectins and VCAM-1 expressed on endothelium of the BM.

CTACK, a skin-associated chemokine that preferentially attracts skin-homing memory T cells

In contrast to naive lymphocytes, memory/effector lymphocytes can access nonlymphoid effector sites and display restricted, often tissue-selective, migration behavior. The cutaneous lymphocyte-associated antigen (CLA) defines a subset of circulating memory T cells that selectively localize in cutaneous sites mediated in part by the interaction of CLA with its vascular ligand E-selectin. Here, we report the identification and characterization of a CC chemokine, cutaneous T cell-attracting chemokine (CTACK). Both human and mouse CTACK are detected only in skin by Southern and Northern blot analyses. Specifically, CTACK message is found in the mouse epidermis and in human keratinocytes, and anti-CTACK mAbs predominantly stain the epithelium. Finally, CTACK selectively attracts CLA+ memory T cells. Taken together, these results suggest an important role for CTACK in recruitment of CLA+ T cells to cutaneous sites. CTACK is predominantly expressed in the skin and selectively attracts a tissue-specific subpopulation of memory lymphocytes.

Rapid evolution of the DNA-binding site in LAGLIDADG homing endonucleases

Sequence analysis of chloroplast and mitochondrial large subunit rRNA genes from over 75 green algae disclosed 28 new group I intron-encoded proteins carrying a single LAGLIDADG motif. These putative homing endonucleases form four subfamilies of homologous enzymes, with the members of each subfamily being encoded by introns sharing the same insertion site. We showed that four divergent endonucleases from the I-CreI subfamily cleave the same DNA substrates. Mapping of the 66 amino acids that are conserved among the members of this subfamily on the 3-dimensional structure of I-CreI bound to its recognition sequence revealed that these residues participate in protein folding, homodimerization, DNA recognition and catalysis. Surprisingly, only seven of the 21 I-CreI amino acids interacting with DNA are conserved, suggesting that I-CreI and its homologs use different subsets of residues to recognize the same DNA sequence. Our sequence comparison of all 45 single-LAGLIDADG proteins identified so far suggests that these proteins share related structures and that there is a weak pressure in each subfamily to maintain identical protein–DNA contacts. The high sequence variability we observed in the DNA-binding site of homologous LAGLIDADG endonucleases provides insight into how these proteins evolve new DNA specificity.

Related homing endonucleases I-BmoI and I-TevI use different strategies to cleave homologous recognition sites

A typical homing endonuclease initiates mobility of its group I intron by recognizing DNA both upstream and downstream of the intron insertion site of intronless alleles, preventing the endonuclease from binding and cleaving its own intron-containing allele. Here, we describe a GIY-YIG family homing endonuclease, I-BmoI, that possesses an unusual recognition sequence, encompassing 1 base pair upstream but 38 base pairs downstream of the intron insertion site. I-BmoI binds intron-containing and intronless substrates with equal affinity but can nevertheless discriminate between the two for cleavage. I-BmoI is encoded by a group I intron that interrupts the thymidylate synthase (TS) gene (thyA) of Bacillus mojavensis s87-18. This intron resembles one inserted 21 nucleotides further downstream in a homologous TS gene (td) of Escherichia coli phage T4. I-TevI, the T4 td intron-encoded GIY-YIG endonuclease, is very similar to I-BmoI, but each endonuclease gene is inserted within a different position of its respective intron. Remarkably, I-TevI and I-BmoI bind a homologous stretch of TS-encoding DNA and cleave their intronless substrates in very similar positions. Our results suggest that each endonuclease has independently evolved the ability to distinguish intron-containing from intronless alleles while maintaining the same conserved recognition sequence centered on DNA-encoding active site residues of TS.

A developmental switch in lymphocyte homing receptor and endothelial vascular addressin expression regulates lymphocyte homing and permits CD4+ CD3- cells to colonize lymph nodes.

IN adult mice, the dominant adhesion molecules involved in homing to lymph nodes are L-selectin homing receptors on lymphocytes and the peripheral lymph node addressins on specialized high endothelial venules. Here we show that, from fetal life through the first 24 hr of life, the dominant adhesion molecules are the mucosal addressin MAdCAM-1 on lymph node high endothelial venules and its counterreceptor, the Peyer's patch homing receptor, integrin alpha 4 beta 7 on circulating cells. Before birth, 40-70% of peripheral blood leukocytes are L-selectin-positive, while only 1-2% expresses alpha 4 beta 7. However, the fetal lymph nodes preferentially attract alpha 4 beta 7-expressing cells, and this can be blocked by fetal administration of anti-MAdCAM-1 antibodies. During fetal and early neonatal life, when only MAdCAM-1 is expressed on high endothelial venules, an unusual subset of CD4 + CD3- cells, exclusively expressing alpha 4 beta 7 as homing receptors, enters the lymph nodes. Beginning 24 hr after birth a developmental switch occurs, and the peripheral node addressins are upregulated on high endothelial venules in peripheral and mesenteric lymph nodes. This switch in addressin expression facilitates tissue-selective lymphocyte migration and mediates a sequential entry of different cell populations into the lymph nodes.

Homing events in the gyrA gene of some mycobacteria.

The A subunit of DNA gyrase in Mycobacterium leprae, unlike its counterpart in Mycobacterium tuberculosis, is produced by protein splicing as its gene, gyrA, harbors a 1260-bp in-frame insertion encoding an intein, a putative homing endonuclease. Analysis of the gyrA locus from different mycobacterial species revealed the presence of inteins in Mycobacterium flavescens, Mycobacterium gordonae and Mycobacterium kansasii but not in 10 other pathogenic or saprophytic mycobacteria. In all four cases where intein coding sequences were found, they were localized in the same position in gyrA, immediately downstream of the codon for the key active-site residue Tyr-130. The intein products were similar, but not identical, in sequence and the splice junctions displayed all the features found in other polypeptides known to be produced by protein splicing from a precursor protein. Paired motifs, found in homing endonucleases encoded by some group I RNA introns, and inteins showing endonuclease activity, were present in the gyrA inteins as were other intein-specific signatures. Some strains of M. flavescens, M. gordonae, and M. kansasii were shown by PCR analysis to have inteinless gyrA genes, in contrast to the situation in M. leprae where all the isolates possessed insertions in gyrA. Sequencing of the corresponding regions revealed that, although the GyrA protein sequence was conserved, the nucleotide sequences differed in gyrA genes with and without inteins, suggesting that the homing endonuclease displays sequence specificity.

Intercellular mobility and homing of an archaeal rDNA intron confers a selective advantage over intron- cells of Sulfolobus acidocaldarius.

Some intron-containing rRNA genes of archaea encode homing-type endonucleases, which facilitate intron insertion at homologous sites in intron- alleles. These archaeal rRNA genes, in contrast to their eukaryotic counterparts, are present in single copies per cell, which precludes intron homing within one cell. However, given the highly conserved nature of the sequences flanking the intron, homing may occur in intron- rRNA genes of other archaeal cells. To test whether this occurs, the intron-containing 23S rRNA gene of the archaeal hyperthermophile Desulfurococcus mobilis, carried on nonreplicating bacterial vectors, was electroporated into an intron- culture of Sulfolobus acidocaldarius. PCR experiments demonstrated that the intron underwent homing and spread through the culture. By using a double drug-resistant mutant of S. acidocaldarius, it was shown that spreading resulted partly from a selective advantage of intron+ cells and partly from intercellular mobility of the intron and homing.

Molecular cloning and functional analysis of SUT-1, a sulfate transporter from human high endothelial venules

High endothelial venules (HEV) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. One of the major characteristics of HEV endothelial cells (HEVEC) is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. Here, we show that HEVEC express two functional classes of sulfate transporters defined by their differential sensitivity to the anion-exchanger inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), and we report the molecular characterization of a DIDS-resistant sulfate transporter from human HEVEC, designated SUT-1. SUT-1 belongs to the family of Na+-coupled anion transporters and exhibits 40–50% amino acid identity with the rat renal Na+/sulfate cotransporter, NaSi-1, as well as with the human and rat Na+/dicarboxylate cotransporters, NaDC-1/SDCT1 and NaDC-3/SDCT2. Functional expression studies in cRNA-injected Xenopus laevis oocytes showed that SUT-1 mediates high levels of Na+-dependent sulfate transport, which is resistant to DIDS inhibition. The SUT-1 gene mapped to human chromosome 7q33. Northern blotting analysis revealed that SUT-1 exhibits a highly restricted tissue distribution, with abundant expression in placenta. Reverse transcription–PCR analysis indicated that SUT-1 and the diastrophic dysplasia sulfate transporter (DTD), one of the two known human DIDS-sensitive sulfate transporters, are coexpressed in HEVEC. SUT-1 and DTD could correspond, respectively, to the DIDS-resistant and DIDS-sensitive components of sulfate uptake in HEVEC. Together, these results demonstrate that SUT-1 is a distinct human Na+-coupled sulfate transporter, likely to play a major role in sulfate incorporation in HEV.

Genetic definition of a protein-splicing domain: Functional mini-inteins support structure predictions and a model for intein evolution

Inteins are protein-splicing elements, most of which contain conserved sequence blocks that define a family of homing endonucleases. Like group I introns that encode such endonucleases, inteins are mobile genetic elements. Recent crystallography and computer modeling studies suggest that inteins consist of two structural domains that correspond to the endonuclease and the protein-splicing elements. To determine whether the bipartite structure of inteins is mirrored by the functional independence of the protein-splicing domain, the entire endonuclease component was deleted from the Mycobacterium tuberculosis recA intein. Guided by computer modeling studies, and taking advantage of genetic systems designed to monitor intein function, the 440-aa Mtu recA intein was reduced to a functional mini-intein of 137 aa. The accuracy of splicing of several mini-inteins was verified. This work not only substantiates structure predictions for intein function but also supports the hypothesis that, like group I introns, mobile inteins arose by an endonuclease gene invading a sequence encoding a small, functional splicing element.

Recurrent invasion and extinction of a selfish gene

Homing endonuclease genes show super-Mendelian inheritance, which allows them to spread in populations even when they are of no benefit to the host organism. To test the idea that regular horizontal transmission is necessary for the long-term persistence of these genes, we surveyed 20 species of yeasts for the ω-homing endonuclease gene and associated group I intron. The status of ω could be categorized into three states (functional, nonfunctional, or absent), and status was not clustered on the host phylogeny. Moreover, the phylogeny of ω differed significantly from that of the host, strong evidence of horizontal transmission. Further analyses indicate that horizontal transmission is more common than transposition, and that it occurs preferentially between closely related species. Parsimony analysis and coalescent theory suggest that there have been 15 horizontal transmission events in the ancestry of our yeast species, through simulations indicate that this value is probably an underestimate. Overall, the data support a cyclical model of invasion, degeneration, and loss, followed by reinvasion, and each of these transitions is estimated to occur about once every 2 million years. The data are thus consistent with the idea that frequent horizontal transmission is necessary for the long-term persistence of homing endonuclease genes, and further, that this requirement limits these genes to organisms with easily accessible germ lines. The data also show that mitochondrial DNA sequences are transferred intact between yeast species; if other genes do not show such high levels of horizontal transmission, it would be due to lack of selection, rather than lack of opportunity.

Localization of α1,3-fucosyltransferase VI in Weibel–Palade bodies of human endothelial cells

Surface glycosylation of endothelial cells is relevant to various processes including coagulation, inflammation, metastasis, and lymphocyte homing. One of the essential sugars involved in these processes is fucose linked α1→3 to N-acetylglucosamine. A family of α1,3-fucosyltransferases (FucTs) called FucT-III, IV, V, VI, VII, and IX is able to catalyze such fucosylations. Reverse transcription–PCR analysis revealed that human umbilical vein endothelial cells express all of the FucTs except FucT-IX. The predominant activity, as inferred by acceptor specificity of enzyme activity in cell lysates, is compatible with the presence of FucT-VI. By using an antibody to recombinant soluble FucT-VI, the enzyme colocalized with β4-galactosyltransferase-1 to the Golgi apparatus. By using a polyclonal antiserum raised against a 17-aa peptide of the variable (stem) region of the FucT-VI, immunocytochemical staining of FucT-VI was restricted to Weibel–Palade bodies, as determined by colocalization with P-selectin and von Willebrand factor. SDS/PAGE immunoblotting and amino acid sequencing of internal peptides confirmed the identity of the antigen isolated by the peptide-specific antibody as FucT-VI. Storage of a fucosyltransferase in Weibel–Palade bodies suggests a function independent of Golgi-associated glycosylation.

Functions of the DNA damage response pathway target Ho endonuclease of yeast for degradation via the ubiquitin-26S proteasome system

Ho endonuclease of Saccharomyces cerevisiae is a homing endonuclease that makes a site-specific double-strand break in the MAT gene in late G1. Here we show that Ho is rapidly degraded via the ubiquitin-26S proteasome system through two ubiquitin-conjugating enzymes UBC2Rad6 and UBC3Cdc34. UBC2Rad6 is complexed with the ring finger DNA-binding protein Rad18, and we find that Ho is stabilized in rad18 mutants. We show that the Ho degradation pathway involving UBC3Cdc34 goes through the Skp1/Cdc53/F-box (SCF) ubiquitin ligase complex and identify a F-box protein, Yml088w, that is required for Ho degradation. Components of a defined pathway of the DNA damage response, MEC1, RAD9, and CHK1, are also necessary for Ho degradation, whereas functions of the RAD24 epistasis group and the downstream effector RAD53 have no role in degradation of Ho. Our results indicate a link between the endonuclease function of Ho and its destruction.

Genetic footprinting with mariner-based transposition in Pseudomonas aeruginosa

The complete DNA sequence of Pseudomonas aeruginosa provides an opportunity to apply functional genomics to a major human pathogen. A comparative genomics approach combined with genetic footprinting was used as a strategy to identify genes required for viability in P. aeruginosa. Use of a highly efficient in vivo mariner transposition system in P. aeruginosa facilitated the analysis of candidate genes of this class. We have developed a rapid and efficient allelic exchange system by using the I-SceI homing endonuclease in conjunction with in vitro mariner mutagenesis to generate mutants within targeted regions of the P. aeruginosa chromosome for genetic footprinting analyses. This technique for generating transposon insertion mutants should be widely applicable to other organisms that are not naturally transformable or may lack well developed in vivo transposition systems. We tested this system with three genes in P. aeruginosa that have putative essential homologs in Haemophilus influenzae. We show that one of three H. influenzae essential gene homologs is needed for growth in P. aeruginosa, validating the practicality of this comparative genomics strategy to identify essential genes in P. aeruginosa.