Sequence-specific interactions of nuclear factors with conserved sequences of human class II major histocompatibility complex genes

All class II major histocompatibility complex genes contain two highly conserved sequences, termed X and Y, within the promoter regions(s), which may have a role in regulation of expression. To study trans-acting factors that interact with these sequences, sequence-specific DNA binding activity has been examined by the gel electrophoresis retardation assay using the HLA-DQ2 beta gene 5' flanking DNA and nuclear extracts derived from various cell types. Several specific protein-binding activities were found using a 45-base-pair (bp) HinfI/Sau96I (-142 to -98 bp) and a 38-bp Sau96I/Sau96I (-97 to -60 bp) fragment, which include conserved sequence X (-113 to -100 bp) and conserved sequence Y (-80 to -71 bp), respectively. Competition experiments, methylation interference analysis, and DNase I foot-printing demonstrated that distinct proteins in a nuclear extract of Raji cells (a human B lymphoma line) bind to sequence X, to sequence Y, and to DNA 5' of the X sequence (termed sequence W). The factor binding site in the W sequence is also found to be conserved among beta-chain genes and is suggested to be a gamma-interferon control region.     

Induction of Rhizobium meliloti nodC expression by plant exudate requires nodD.

The soil bacterium Rhizobium meliloti invades and establishes a symbiosis with host plants such as alfalfa. Bacterial nodulation (nod) genes are required for this invasion, but their mechanism of action and the timing of their expression are not known. We have used translational lacZ fusions to monitor expression of nodD and nodC, which are located in the cluster of four nod genes on the R. meliloti megaplasmid (pSym). nodD is expressed at comparable levels by broth-grown bacterial cells and by cells exposed to exudates from aseptically grown plants. Activity of the nodC-lacZ protein fusion in broth-grown bacterial cells is very low. nodC-lacZ activity is increased approximately equal to 30-fold by plant exudate when nodD is expressed at a high level but not when nodD expression is low. Both fusions show differences in expression when borne on inc-P vectors as compared to when located on the pSym megaplasmid. nodD expression from vector-borne copies of the nod segment and response of nodC to plant exudate appear to require additional loci on the megaplasmid. Our results suggest that regulation of bacterial nod gene expression is an important control mechanism early in the symbiosis, and that the biochemical nature of some nod gene products may be cryptic except in cells grown in the presence of plant exudate.     

Rhizobium meliloti has three functional copies of the nodD symbiotic regulatory gene.

We have identified two Rhizobium meliloti genes (nodD2 and nodD3) that are highly homologous and closely linked to the regulatory gene nodD (nodD1). R. meliloti strains containing mutations in the three nodD genes in all possible combinations were constructed and their nodulation phenotypes were assayed on Medicago sativa (alfalfa) and Melilotus alba (sweet clover). A triple nodD1-nodD2-nodD3 mutant exhibited a Nod- phenotype on alfalfa and sweet clover, indicating that nodD is an essential nodulation gene in R. meliloti. A nodD2 mutant exhibited no discernable defect in nodulation and nodD3 mutants exhibited a delayed nodulation phenotype of 2-3 days when inoculated onto either host. Alfalfa nodules elicited by a nodD1 mutant appeared 5-6 days after wild-type nodules, and sweet clover nodules elicited by a nodD1 mutant appeared 2-3 days after wild-type nodules. nodD1-nodD2 double mutants formed nodules with the same delay as single nodD1 mutants on both hosts. nodD2-nodD3 double mutants elicited sweet clover nodules at the same rate as single nodD3 mutants, but this same double mutant was slightly more delayed in alfalfa nodule formation than the nodD3 mutant. The nodD1-nodD3 mutant exhibited an extremely delayed nodulation phenotype on alfalfa and elicited no nodules on sweet clover. These experiments indicate that nodD1 and nodD3 have equivalent roles in nodulating sweet clover but that nodD1 plays a more important role than nodD3 in eliciting nodules on alfalfa. The nodD2 gene appears to have some effect on alfalfa nodulation and none on sweet clover. Our results indicate that R. meliloti has three functional nodD genes that modulate the nodulation process in a host-specific manner.     

Interspecies homology of nitrogenase genes.

Cloned nitrogen fixation (nif) genes from Klebsiella pneumoniae hybridize to DNA from 19 out of 19 widely divergent nitrogen-fixing bacterial strains but do not hybridize to DNA from 10 different non-nitrogen-fixing species. K. pneumoniae nif DNA fragments that hybridize to DNA from other species contain part of the three structural genes that code for nitrogenase polypeptides. We have utilized this homology to clone an EcoRI restriction endonuclease fragment from Rhizobium meliloti that hybridizes to the K. pneumoniae nif structural genes. Some of the species whose DNA hybridizes with K. pneumoniae nif DNA have been postulated to have diverged from K. pneumoniae 3 x 10(9) years ago. Nitrogenase genes are the only known example of such highly conserved prokaryotic translated genes. Nitrogenase genes are either extraordinarily conserved in evolution or have been exchanged between different nitrogen-fixing species relatively recently in evolutionary time.     

Biosynthesis of Rhizobium meliloti lipooligosaccharide Nod factors: NodA is required for an N-acyltransferase activity.

Rhizobium bacteria synthesize N-acylated beta-1,4-N-acetylglucosamine lipooligosaccharides, called Nod factors, which act as morphogenic signal molecules to legume roots during development of nitrogen-fixing nodules. The biosynthesis of Nod factors is genetically dependent upon the nodulation (nod) genes, including the common nod genes nodABC. We used the Rhizobium meliloti NodH sulfotransferase to prepare 35S-labeled oligosaccharides which served as metabolic tracers for Nod enzyme activities. This approach provides a general method for following chitooligosaccharide modifications. We found nodAB-dependent conversion of N-acetylchitotetraose (chitotetraose) monosulfate into hydrophobic compounds which by chromatographic and chemical tests were equivalent to acylated Nod factors. Sequential incubation of labeled intermediates with Escherichia coli containing either NodA or NodB showed that NodB was required before NodA during Nod factor biosynthesis. The acylation activity was sensitive to oligosaccharide chain length, with chitotetraose serving as a better substrate than chitobiose or chitotriose. We constructed a putative Nod factor intermediate, GlcN-beta 1,4-(GlcNAc)3, by enzymatic synthesis and labeled it by NodH-mediated sulfation to create a specific metabolic probe. Acylation of this oligosaccharide required only NodA. These results confirm previous reports that NodB is an N-deacetylase and suggest that NodA is an N-acyltransferase.     

Cloning and complete nucleotide sequence of human 5''-alpha-globin gene.

We have cloned one of the two human alpha-globin genes and report its complete nucleotide sequence. The gene is 832 base pairs (bp) long from the 5'-cap site to the 3'-polyadenylylation site. The amino acid coding sequences are separated into three segments (exons) by two short (117 and 140 bp) intervening sequences. Highly conserved regions are identified in the 5'-flanking region, intron-exon junctions, and 3' noncoding regions that may have functional significance.     

Rhizobium NodB protein involved in nodulation signal synthesis is a chitooligosaccharide deacetylase.

The common nodulation genes nodABC are conserved in all rhizobia and are involved in synthesis of a lipooligosaccharide signal molecule. This bacterial signal consists of a chitooligosaccharide backbone, which carries at the nonreducing end a fatty acyl chain. The modified chitooligosaccharide molecule triggers development of nodules on the roots of the leguminous host plant. To elucidate the specific role of the NodB protein in nodulation factor synthesis, we have purified recombinant NodB and determined its biochemical role by direct assays. Our data show that the NodB protein of Rhizobium meliloti deacetylates the nonreducing N-acetylglucosamine residue of chitooligosaccharides. The monosaccharide N-acetylglucosamine is not deacetylated by NodB. In the pathway of Nod factor synthesis, deacetylation at the nonreducing end of the oligosaccharide backbone may be a necessary requirement for attachment of the fatty acyl chain.     

Biosynthesis of lipooligosaccharide nodulation factors: Rhizobium NodA protein is involved in N-acylation of the chitooligosaccharide backbone.

Rhizobium meliloti interacts symbiotically with alfalfa by forming root nodules in which the bacteria fix nitrogen. The Rhizobium nodulation genes nodABC are involved in the synthesis of lipooligosaccharide symbiotic signal molecules, which are mono-N-acylated chitooligosaccharides. These bacterial signals elicit nodule organogenesis in roots of legumes. To elucidate the role of the NodA protein in lipooligosaccharide biosynthesis, we prepared a radiolabeled tetrasaccharide precursor carrying an amino group as a potential attachment site for N-acylation at the nonreducing glucosamine residue. Various criteria demonstrate that NodA is involved in the attachment of a fatty acyl chain to this tetrasaccharide precursor, yielding a biologically active nodulation factor.     

Conservation of nodulation genes between Rhizobium meliloti and a slow-growing Rhizobium strain that nodulates a nonlegume host

Parasponia, a woody member of the elm family, is the only nonlegume genus whose members are known to form an effective nitrogen-fixing symbiosis with a Rhizobium species. The bacterial strain RP501 is a slow-growing strain of Rhizobium isolated from Parasponia nodules. Strain RP501 also nodulates the legumes siratro (Macroptilium atropurpureum) and cowpea (Vigna unguiculata). Using a cosmid clone bank of RP501 DNA, we isolated a 13.4-kilobase (kb) EcoRI fragment that complemented insertion and point mutations in three contiguous nodulation genes (nodABC) of Rhizobium meliloti, the endosymbiont of alfalfa (Medicago sativa). The complemented R. meliloti nod mutants induced effective nitrogen-fixing nodules on alfalfa seedlings but not on siratro, cowpeas, or Parasponia. The cloned RP501 nodulation locus hybridized to DNA fragments carrying the R. meliloti nodABC genes. A 3-kb cluster of Tn5 insertion mutations on the RP501 13.4-kb EcoRI fragment prevented complementation of R. meliloti nodABC mutations.     

Territorial limits and functional anatomy of the simian virus 40 replication origin.

The region at and near the simian virus 40 (SV40) DNA replication origin contains a series of palindromes, a 17-base pair (bp) A + T-rich sequence, three copies of a 21-bp repeat, and two copies of a 72-bp repeat. We have constructed a series of recombinant plasmids containing sequential deletions at the region of SV40 DNA replication origin starting from the end near the repeats. These deletions were introduced by using in vitro and in vivo techniques. The relative replication efficiency of these recombinant plasmids were directly assayed in COS-1 monkey kidney cells capable of providing the tumor antigen necessary for the replication of these molecules. Recombinants lacking both copies of the 72-bp repeat did not exhibit any reduction in replication efficiency. Recombinants lacking the 21-bp repeats showed decreased replication efficiency; the reduction in replication efficiency was proportional to the number of copies of the 21-bp repeat deleted in these recombinants. A recombinant retaining the palindromes at the region of SV40 DNA replication but lacking the A + T-rich sequence and the repeats failed to replicate. Based on these results, the SV40 DNA replication origin is subdivided into two regions, and their boundaries are defined. One of these two regions is a core region containing the 17-bp, 15-bp, and 27-bp palindromes and, quite likely, the 17-bp A + T-rich sequence which are necessary for replication. The other is an auxiliary region that consists of the 21-bp repeats and has a dose-dependent enhancement effect on replication efficiency.     

Nucleotide sequence analysis of the chicken c-myc gene reveals homologous and unique coding regions by comparison with the transforming gene of avian myelocytomatosis virus MC29, delta gag-myc.

Myelocytomatosis virus MC29 is a defective avian retrovirus with a hybrid transforming gene (delta gag-myc) consisting of a 1,358-base pair (bp) sequence from the retroviral gag gene and a 1,568-bp sequence (v-myc) shared with a cellular locus, termed c-myc. We have subjected to sequence analysis 2,735 bp of the cloned c-myc gene, which includes the v-myc-related region of 1,568 bp, an intervening sequence of 971 bp, and unique flanking sequences of 45 bp and 195 bp at the 5' and 3' ends, respectively. Analysis of the genetic information and alignment of the c-myc sequence with the known sequence of MC29 indicates that: (i) the two myc sequences share the same reading frame, including the translational termination signal; (ii) there are nine nucleotide changes between c-myc and v-myc that correspond to seven amino acid changes; (iii) the 971-bp intervening sequence of c-myc can be defined as an intron by consensus splice signals; (iv) the unique 5' sequence of c-myc could either extend its reading frame beyond the homology with v-myc or could be an intron because its junction with the myc region of the locus is a canonical 3' splice-acceptor site; (v) the v-myc contains 10 nucleotides at its 5' end not shared with the c-myc analyzed here and also not with known gag genes, probably derived from an upstream exon; and (vi) the c-myc locus can generate a mRNA whose termination signals have been identified to be located 83 bp and 119 bp from the point of divergence between the v-myc and c-myc. We conclude that the gene of the c-myc locus of the chicken and the onc gene of MC29 share homologous myc regions and differ in unique 5' coding regions and we speculate, on this basis, that their protein products may have different functions. The hybrid onc gene of MC29 must have been generated from the c-myc gene by deletion of the 5' cellular coding sequence, followed by substitution with the 5' region of the viral gag gene.     

In vitro sulfotransferase activity of Rhizobium meliloti NodH protein: lipochitooligosaccharide nodulation signals are sulfated after synthesis of the core structure.

The Rhizobium common nod gene products NodABC are involved in the synthesis of the core lipochitooligosaccharide (Nod factor) structure, whereas the products of the host-specific nod genes are necessary for diverse structural modifications, which vary in different Rhizobium species. The sulfate group attached to the Rhizobium meliloti Nod signal is necessary for activity on the host plant alfalfa, while its absence renders the Nod factor active on the non-host plant vetch. This substituent is therefore a major determinant of host specificity. The exact biosynthetic pathway of Nod factors has not been fully elucidated. In particular, it is not known why some chemical modifications are introduced with high fidelity whereas others are inaccurate, giving rise to a family of different Nod factor structures produced by a single Rhizobium strain. Using protein extracts and partially purified recombinant NodH protein obtained from Escherichia coli expressing the R. meliloti nodH gene, we demonstrate here NodH-dependent in vitro sulfotransferase activity. Kinetic analyses with Nod factors, chitooligosaccharides, and their deacetylated derivatives revealed that Nod factors are the preferred substrate for the sulfate transfer. Moreover, the tetrameric Nod factor, NodRm-IV, was a better substrate than the trimer, NodRm-III, or the pentamer, NodRm-V. These data suggest that the core lipochitooligosaccharide structure must be synthesized prior to its host-specific modification with a sulfate group. Since in R. meliloti tetrameric Nod factors are the most abundant and the most active ones, high affinity of NodH for the appropriate tetrameric substrate guarantees its modification and thus contributes to the fidelity of host-specific behavior.     

Multiple regulatory regions on the 5' side of the mouse E alpha gene

The function of the 5'-flanking region of the mouse major histocompatibility complex gene Ed alpha has been studied by deletion analysis with the chloramphenicol acetyltransferase gene as a transient expression marker in various cell lines. This analysis reveals the presence of several control regions on the 5' side of the gene. Sequences between base pair (bp) -873 and bp -353 have a negative function in human and mouse fibroblasts but not in the mouse macrophage line WEHI-3. Additional positive and negative elements have been mapped between bp -353 and bp -38. A gamma-interferon response region has been also identified within that sequence. the 5' and 3' boundaries of the gamma-interferon response region have been located between bp -164 and bp -43. Inducible human cell lines showed the same gamma-interferon response region endpoints with the mouse cell line WEHI-3. A DNA fragment spanning the equivalent region of the mouse Ed beta gene confers gamma-interferon inducibility to the simian virus 40 and alpha-globin promoters in an orientation-independent manner. We further provide evidence that the conserved sequence motifs on the 5' side of all major histocompatibility complex class II genes are indispensable for gamma-interferon induction.