Soybean leghemoglobin gene family: normal, pseudo, and truncated genes.

Leghemoglobin (Lb) genes in soybean represent a small family of closely related genes. Three Lb sequences isolated from a genomic library were analyzed at the nucleotide sequence level. A Lb gene present on an 11.5-kilobase (kb) EcoRI genomic fragment spans approximately 1,200 nucleotides and is interrupted at amino acid positions 32 to 33, 68 to 69, and 103 to 104. The intervening sequences, as well as the 5' and 3' flanking regions of this gene, contain the consensus sequences found in other eukaryotic genes. The length of the 5'-untranslated region is 49 bases as determined by nuclease S1 mapping. R-loop analysis of the DNA from the recombinant phage containing the 11.5-kb EcoRI genomic fragment showed that another Lb gene is located 2.5 kb away. The nucleotide sequence of the second gene showed that this gene is incomplete, containing only exons 3 and 4. The deduced amino acid sequence of this gene, although showing 76% homology with the corresponding region of the other Lb gene, is not represented in any of the known Lb proteins. Both genes are oriented in the same direction with respect to the coding strand. Analysis of the sequence present on a second genomic clone containing a 4.2-kb EcoRI fragment revealed a truncated Lb gene showing homology with the last exon and the noncoding region at the 3' end of the two other Lb genes.     

Bacillus stearothermophilus contains a plasmid-borne gene for alpha-amylase.

The gene for thermostable alpha-amylase from the thermophilic bacterium Bacillus stearothermophilus has been cloned and expressed in Escherichia coli. Each alpha-amylase-producing colony contained at least a 9.7-kilobase-pair (kb) chimeric plasmid composed of the vector pBR322 and a common 5.4-kb HindIII fragment of DNA. B. stearothermophilus contains four plasmids with sizes from 12 kb to over 108 kb. Restriction endonuclease analysis of these naturally occurring plasmids showed they also contain a 5.4-kb HindIII fragment of DNA. Cloning experiments with the four plasmids yielded alpha-amylase-producing E. coli that contained the same 9.7-kb chimeric plasmid. Restriction endonuclease analysis and further recombinant DNA experiments identified a 26-kb plasmid that contains the gene for alpha-amylase. A spontaneous mutant of B. stearothermophilus unable to produce alpha-amylase was missing the 26-kb plasmid but contained a 20-kb plasmid. A 6-kb deletion within the region of the 5.4-kb HindIII fragment yielded the 20-kb plasmid unable to code for alpha-amylase. A nick-translated probe for the alpha-amylase coding region did not hybridize to either plasmid or total cellular DNA from this mutant strain of B. stearothermophilus. These results demonstrate the gene for alpha-amylase is located exclusively on a 26-kb plasmid in B. stearothermophilus with no genetic counterpart present on the chromosome.     

Gene conversion-like events cause steroid 21-hydroxylase deficiency in congenital adrenal hyperplasia.

Genomic DNAs from twelve Japanese patients with steroid 21-hydroxylase [21-OHase; steroid 21-monooxygenase; steroid, hydrogen-donor:oxygen oxidoreductase (21-hydroxylating); EC 1.14.99.10] deficiency were analyzed by Southern blot hybridization. A 3.7-kilobase (kb) Taq I and a 1.7-kb Pvu II restriction endonuclease fragment that correspond to a 21-OHase B gene were absent from the DNA of two unrelated patients with the salt-wasting form of the disease. However, a 10.5-kb Bgl II fragment corresponding to the region encompassing the 21-OHase B gene was still present in these two patients. The genes encoding 21-OHase were cloned from one of these two patients, who was homozygous by descent for HLA-A26;B39;C4A3;C4B1;DR4. Restriction endonuclease mapping as well as partial nucleotide sequencing analysis revealed that the 21-OHase B gene of the patient has been converted to the pseudogene, 21-OHase A, as far as the critical 0.5-kb sequence was concerned. Thus, the defect was due to both chromosomes each carrying two copies of 21-OHase A pseudogene and lacking functional 21-OHase B gene.     

Polymorphic restriction endonuclease sites linked to the HLA-DR alpha gene: localization and use as genetic markers of insulin-dependent diabetes.

Polymorphic restriction endonuclease sites within the HLA-DR alpha gene have been defined, localized, and used as genetic markers in the analysis of susceptibility to insulin-dependent diabetes mellitus (IDDM). Hybridization of Bgl II-digested human genomic DNA with a cDNA clone for the HLA-DR alpha chain (pDR alpha-1) has revealed three allelic restriction fragment lengths: 3.8 kilobase pairs (kb), 4.2 kb, and 4.5 kb. Hybridization of EcoRV-digested human genomic DNA with the same probe has revealed two allelic polymorphic restriction fragment lengths: 9.2 kb and 13.0 kb. By analysis of double digests of genomic DNA from individuals homozygous for each of the allelic variants, the polymorphic restriction sites were found to be clustered near the 3' end of the HLA-DR alpha gene. The observed correlations of DR alpha Bgl II restriction site variants with serologically determined DR specificities suggest linkage disequilibrium between the DR alpha and DR beta loci. The 3.8-kb fragment is correlated with the DR1 type (Pc = 4.4 X 10(-4)); and the 4.2-kb fragment, with a subset (B8,DR3) of the DR3 type (Pc = 5.1 X 10(-4)) and with the DR6 type. The segregation pattern of HLA-DR alpha polymorphic Bgl II restriction fragments was analyzed in six IDDM families. The observed association of IDDM with the Bgl II 4.2-kb DR alpha restriction variant is higher than with existing serological markers and supports the utility of this approach in elucidating IDDM inheritance.     

Intracisternal A-particle genes: identification in the genome of Mus musculus and comparison of multiple isolates from a mouse gene library.

The genome of Mus musculus contains multiple copies (500 -1000) of DNA sequences related to the 35S RNA of intracisternal type A particles (IAPs). Using labeled IAP RNA as a probe in blot-hybridization experiments, we have identified a characteristic electrophoretic pattern of reactive fragments generated by restriction endonuclease cleavage of mouse DNA. From the genomic blots, we deduced a composite restriction map for a 6.5- to 7-kilobase (kb) DNA region containing sequences homologous to the IAP RNA. Units of this type appeared to be interspersed without obvious regularity in nonhomologous flanking regions. A 5.2-kb segment of this unit was inserted directly into plasmid pBR322 from HindIII/EcoRI digest of mouse DNA. The fragment was cloned and then labeled by nick-translation and used to scan a mouse embryo gene library (average 16-kb inserts in lambda Charon 4A); 1% of the library samples hybridized, confirming the extensive reiteration of IAP genetic units. Among six different library isolates containing 6.5- to 7-kb IAP units, some restriction sites were highly conserved whereas others varied in both occurrence and position. Despite this variation, heteroduplexes between the individual isolates showed continuous IAP homology regions of 7 kb. No flanking region homologies were seen in this limited sample. Some evidence suggests that mouse DNA may contain other dispersed sequence elements related to but smaller than the genetic unit defined above.     

Structure of genes for human growth hormone and chorionic somatomammotropin.

A 2.6-kilobase (kb) EcoRI restriction endonuclease fragment containing human growth hormone (hGH; somatotropin) gene sequences and a 2.8-kb EcoRI fragment containing human chorionic somatomammotropin (hCS; choriomammotropin) gene sequences have been identified by hybridization to cloned cDNA. Human DNA was cleaved with EcoRI and fractionated by preparative agarose gel electrophoresis; DNA in the size range 2--3 kb was ligated to lambda gt WES.lambda B DNA and viable recombinant bacteriophage were recovered by in vitro packaging. After infection of Escherichia coli and screening of phage plaques, single isolates of hGH and hCS gene sequences were obtained. Restriction endonuclease mapping showed that the hGH gene contains three intervening sequences interrupting the coding sequence. Partial DNA sequence analysis of the hGH gene, obtained by the chain termination method, confirmed the location of the intervening sequences and the identity of the fragment.     

Gene conversion in salt-losing congenital adrenal hyperplasia with absent complement C4B protein

Two of four siblings expressed the salt-losing form of congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH) and had identical human lymphocyte antigen (HLA) and complement C4 (fourth component of complement) types (HLA-A3,C4,B35,C4A3,C4BQO,DR1/A2,C-,B18,C4A3, C4BQO,DR6). The father and one unaffected sibling were heterozygous carriers of CAH, as determined by a 30-min iv ACTH stimulation test and HLA typing. In addition, the iv ACTH stimulation test revealed that the mother and the other unaffected sibling also carried an allele for an attenuated form of CAH. Restriction endonuclease digests of genomic DNA obtained from members of this family and from normal unrelated subjects were hybridized with cDNA probes encoding human 21-hydroxylase and C4. With the 21-hydroxylase probe, Southern blots prepared from control DNA samples revealed two major restriction fragments in each of four restriction endonuclease digests; TaqI produced major bands at 3.7 and 3.2 kilobases (kb), KpnI at 4.0 and 2.9 kb, EcoRI at 18 and 13 kb, and BglII at 15 and 12.5 kb. Southern blots prepared from DNA of the two patients lacked the 3.7-kb TaqI and 2.9-kb KpnI fragments, but had increased hybridization intensity (relative to control DNA samples) in the 3.2-kb TaqI and 4.0-kb KpnI fragments. By contrast, blots with EcoRI or BglII had two large hybridization fragments not different from control DNA samples. These data indicate the presence of two different 21-hydroxylase genes. Additional mapping studies revealed that the two genes had the restriction pattern of the inactive 21-hydroxylase gene. When genomic DNA that had been isolated from all members of this family and from normal subjects was hybridized with the human C4 cDNA probe, the restriction fragment hybridization patterns for all four endonuclease digests were similar in the two groups. Hence, our results suggest that the 21-hydroxylase deficiency of our patients is due to conversion of the active 21-hydroxylase gene to the inactive gene. This gene conversion was associated with absence of functional C4B protein, without any detectable alterations in the restriction fragment pattern of the C4 genes.     

Mitochondrial DNA rearrangement associated with fertility restoration and cytoplasmic reversion to fertility in cytoplasmic male sterile Phaseolus vulgaris L

Restoration of pollen fertility to cytoplasmic male sterile (CMS) Phaseolus vulgaris by a nuclear restorer gene provides a system for studying nuclear-cytoplasmic interactions. Introduction of a nuclear restorer gene to this CMS line of P. vulgaris (CMS-Sprite) results in a mitochondrial genome rearrangement similar to that observed upon spontaneous cytoplasmic reversion to fertility. Three spontaneous heritable cytoplasmic revertants were derived from CMS-Sprite. Five fully fertile restored lines were also produced by using restorer line R-351 (BC₃F₃ populations). Comparison of the mitochondrial DNA restriction patterns of CMS-Sprite, the three fertile revertants, and the five restored lines revealed loss of a 6.0-kilobase (kb) Pst I fragment in all restored and revertant lines. Southern hybridizations with a 1.3-kb BamHI clone, internal to the 6.0-kb Pst I fragment, as a probe revealed two configurations of 6.0-kb homologous sequences in the sterile cytoplasm; one of the configurations was lost upon reversion or restoration. Mitochondrial DNA rearrangement has thus been observed upon restoration by a nuclear restorer gene in this CMS system.     

HLA-linked congenital adrenal hyperplasia results from a defective gene encoding a cytochrome P-450 specific for steroid 21-hydroxylation.

We have determined the molecular genetic basis of congenital adrenal hyperplasia due to 21-hydroxylase (21-OHase) deficiency. This common disorder of cortisol biosynthesis is HLA-linked. The haplotype HLA-(A3);Bw47;DR7 is strongly associated with 21-OHase deficiency and always carries a null allele at the locus encoding the C4A (Rodgers) form of the fourth component (C4) of complement. It seemed likely that this haplotype carries a deletion encompassing the genes encoding both C4A and 21-OHase. We hypothesized that the HLA-linked defect involved a structural gene for the adrenal microsomal cytochrome P-450 specific for steroid 21-hydroxylation. Using a plasmid with a 520-base-pair bovine adrenal cDNA insert encoding the middle third of the cytochrome P-450 polypeptide, we compared hybridization patterns in DNA from normal and 21-OHase-deficient individuals. Normal human DNA yielded two fragments that hybridized with the probe after digestion with either restriction endonuclease EcoRI [12- and 14-kilobase (kb) fragments] or Taq I (3.7 and 3.2 kb). One of these bands (the first mentioned in each digest) was absent in DNA from a cell line derived from a patient homozygous for HLA-Bw47. DNA from six unrelated patients homozygous for 21-OHase deficiency who were heterozygous for HLA-Bw47 yielded diminished relative intensity of the 3.7-kb Taq I band in five patients, consistent with a heterozygous deletion, and complete disappearance of the 3.7-kb band in one. This deletion segregated with HLA-Bw47 in a large pedigree carrying 21-OHase deficiency and HLA-Bw47. Thus, 21-OHase deficiency sometimes results from the deletion of a specific cytochrome P-450 gene and sometimes, presumably, from smaller mutations. This gene is probably located very near the C4A gene.     

Structure and function of a chloroplast DNA replication origin of Chlamydomonas reinhardtii

Chloroplast DNA replication in Chlamydomonas reinhardtii is initiated by the formation of a displacement loop (D-loop) at a specific site. One D-loop site with its flanking sequence was cloned in recombinant plasmids SC3-1 and R-13. The sequence of the chloroplast DNA insert in SC3-1, which includes the 0.42-kilobase (kb) D-loop region, as well as 0.2 kb to the 5′ end and 0.43 kb to the 3′ end of the D-loop region, was determined. The sequence is A+T-rich and contains four large stem-loop stuctures. An open reading frame potentially coding for a polypeptide of 136 amino acids was detected in the D-loop region. One stem-loop structure and two back-to-back prokaryotic-type promoters were mapped within the open reading frame. The 5.5-kb EcoRI fragment cloned in R-13 contains the 1.05-kb SC3-1 insert and its flanking regions. A yeast autonomously replicating (ARS) sequence and an ARC sequence, which promotes autonomous replication in Chlamydomonas, have been mapped within the flanking regions [Vallet, J.-M. & Rochaix, J.-D. (1985) Curr. Genet. 9, 321-324]. Both R-13 and SC3-1 were active as templates in a crude algal preparation that supports DNA synthesis. In this in vitro system, chloroplast DNA synthesis initiated near the D-loop site.     

Structural organization of the DR subregion of the human major histocompatibility complex.

Two clusters of overlapping cosmid and lambda phage clones comprising 205 kilobases (kb) have been isolated from the DR subregion of the human major histocompatibility complex from a DR4 haplotype. A single DR alpha and three DR beta genes were identified. In one cluster (135 kb), the DR alpha gene is 90 kb distant from the DR beta gene encoding a molecule that carries the MT3 serological specificity. In the second cluster (70 kb), the DR beta gene determining the DR4 specificity is located 22 kb apart from a DR beta pseudogene (DR beta psi). A 3- to 4-kb sequence located at the 5' end of the DR beta (MT3) gene is common to all three DR beta-chain genes. In addition, three more copies of this sequence are spaced between the DR alpha and the DR beta (MT3) genes in the first cluster and one of these, at least, is associated with a DR beta 1 exon, suggesting that additional genes could be encoded in this region and that multiple duplication events have led to its evolution.     

Putative actin genes in the macronucleus of Oxytricha fallax.

Previous work has shown that the macronuclear DNA of the hypotrichous ciliate Oxytricha fallax is arranged as short achromosomal pieces, 22 to 0.5 kilobase pairs (kb) in length. Micronuclear DNA has a typical chromosomal organization. Macronuclear DNA is derived from micronuclear DNA through a process of polytene chromosome fragmentation with a resultant decrease in DNA sequence complexity. Three putative actin genes have been identified in macronuclear DNA by using a cloned yeast actin gene as a hybridization probe. A restriction fragment of the yeast gene containing both actin coding and noncoding DNA hybridizes strongly to two macronuclear DNA pieces, 1.6 and 1.4 kb in length, and weakly to a 1.2-kb piece. The entire 1.6-kb piece has been cloned in plasmid pBR322 and the resulting recombinant plasmid has been designated pOfACT(1.6). The 1.6-kb pOfACT(1.6) insert hybridizes only to those restriction fragments of the yeast actin gene containing actin coding sequences. When hybridized to macronuclear DNA under conditions that allow the yeast probe to hybridize to all three macronuclear pieces, the pOfACT(1.6) insert hybridizes only to the 1.6-kb piece. Under less stringent conditions the insert also hybridizes to the 1.4-kb piece, but it shows no hybridization to the 1.2-kb DNA. The three macronuclear pieces homologous to the yeast actin gene thus differ in sequence and are interpreted as a related family of actin genes. Each of these pieces could accommodate an actin coding sequence, which in yeast, Dictyostelium discoideum, and Drosophila melanogaster is 1.1 kb, and an additional 0.1-0.5 kb of noncoding DNA.     

Isolation of the origin of replication associated with the amplified Chinese hamster dihydrofolate reductase domain.

Autoradiography of restriction digests of DNA labeled in early S phase indicates that replication of the amplified dihydrofolate reductase (DHFR) domain of methotrexate-resistant CHOC 400 cells initiates within a 6.1-kilobase pair (kb) EcoRI-doublet located on the 3' side of the DHFR gene. To localize the DHFR origin fragment, synchronized CHOC 400 cells were either pulse labeled with [3H]thymidine in vivo or permeabilized and incubated with [32P]dATP under conditions that support limited chromosomal DNA replication. The temporal order of replication of amplified fragments was determined by hybridization of the in vivo or in vitro replication products to cloned fragments spanning the earliest-replicating portion of the DHFR domain. At the G1/S boundary, the labeled products derived from the replication of amplified sequences, either in whole or permeabilized cells, are distributed about an amplified 4.3-kb Xba I fragment that maps 14 kb downstream from the DHFR gene. As cells progress through the S phase, bidirectional replication away from this site is observed. These studies indicate that the 4.3-kb Xba I fragment contains the origin of replication associated with the amplified DHFR domain.     

The ets sequence from the transforming gene of avian erythroblastosis virus, E26, has unique domains on human chromosomes 11 and 21: both loci are transcriptionally active.

Human DNA segments homologous to the ets region from the transforming gene of avian erythroblastosis virus, E26, were molecularly cloned and shown to be closely related to the viral equivalent by hybridization and partial sequence analysis. The transforming gene of E26 has a tripartite origin with the structure delta gag [1.2 kilobases (kb) from the viral gag gene]-myb(0.9 kb from the chicken myb gene)-ets (1.6 kb from the chicken ets gene). Human ets DNA is located on two distinct human chromosomes. The human ets-1 locus on chromosome 11 encodes a single mRNA of 6.8 kb; the second locus, ets-2 on chromosome 21, encodes three mRNAs of 4.7, 3.2, and 2.7 kb. The ets-related sequences of human DNA on chromosomes 11 and 21 are discontiguous, except for a small overlap region encoding 14 amino acids, where 12 are conserved between these two loci. By contrast, the chicken homolog has contiguous ets-1 and ets-2 sequences and is primarily expressed in normal chicken cells as a single 7.5-kb mRNA. We conclude that the ets sequence shared by the virus, the chicken, and humans is likely to contain at least two dissociable functional domains, ets-1 and ets-2. Thus, the tripartite transforming gene of E26 includes four distinct domains that may be functionally relevant for the transforming function of the virus (delta gag, myb, ets-1, and ets-2).     

Close correlation between restriction fragment length polymorphism of the L-MYC gene and metastasis of human lung cancer to the lymph nodes and other organs.

Restriction length fragment polymorphism of the L-MYC gene was examined in DNAs from lung cancer tissues and normal tissues of 51 Japanese patients with lung cancer. In individual patients, no difference was seen between the restriction length fragments of the two alleles of L-MYC [6-kilobase (kb) and 10-kb fragments in EcoRI digests] in lung cancer tissues and normal tissues. But a striking correlation was found between the restriction length fragment polymorphism pattern of L-MYC and the extent of metastasis, particularly to the lymph nodes at the time of surgery: Patients with only the L band (10 kb) had few lymph node metastatic lesions, whereas patients with either the S band (6 kb) or the S and L bands almost always had lymph node metastatic lesion. A similar correlation was found between the presence of the S band and metastases to other organs. This correlation was particularly marked in cases of adenocarcinoma. These results indicate a clear genetic influence on metastases and a consequent poor prognosis for certain patients of lung cancer; L-MYC restriction length fragment polymorphism is thus shown to be a useful marker for predicting the metastatic potential of human lung cancer.     

Analysis of gonadotropin-releasing hormone gene structure in families with familial central precocious puberty and idiopathic hypogonadotropic hypogonadism

We examined the GnRH gene structure in a family with familial central precocious puberty (eight members, four affected) and a family with idiopathic hypogonadotropic hypogonadism (eight members, three affected) using Southern blot analysis and sequencing of cloned polymerase chain reaction products. Genomic DNA samples were digested with restriction enzymes and hybridized to the human placental GnRH cDNA probe. BamHI digests revealed 6.5- and 2.7-kilobase (kb) bands; BglII, 6.0- and 4.0-kb bands; Ncol, 8.0- and 3.5-kb bands; Pstl, 4.2-kb, 2.8-kb, 1.3-kb and 950-basepair bands; XbaI, 6.5- and 5.0-kb bands. These sizes were the same as those found by this analysis in normal individuals. All family members with familial central precocious puberty or idiopathic hypogonadotropic hypogonadism showed the same size bands, except for one unaffected member of the family with idiopathic hypogonadotropic hypogonadism who had an additional band at 5.5 kb after digestion with NcoI, which is thought to be a rare polymorphism. Sequencing of exon 2 of the GnRH gene from these families, including the exon-intron borders, revealed a polymorphism in the signal sequence of GnRH that predicts an amino acid change from tryptophan (nucleotide sequence: TGG) to serine (TCG) at the -8 position of the GnRH preprohormone. Although this polymorphism did not cosegregate with the clinical disorder in either family, this novel polymorphism may prove useful in the evaluation of linkage to the GnRH gene in other families with pubertal disorders. No other nucleotide sequence abnormality was found in 1.2 kb of the 5' flanking region or the four exons and their splice sites.     

The modification and expression of 21-hydroxylase gene in normal human adrenal gland and adrenal cancer.

Genomic DNA and total RNA from three adrenal cancer tissues were analyzed by hybridization with human 21-hydroxylase gene. Southern blot analysis with restriction enzyme Eco RI revealed major fragments at 18, 13 and 9 kilobase (kb) in normal adrenal glands. In two of three adrenal cancers, however, the band at 18 kb was either absent or decreased and the 9 kb fragment showed an increase in its intensity. Normal liver, kidney and leucocytes has only 18 and 13 kb while lacking the 9 kb fragment. Using Taq I, Bam HI or Bgl II, we found no difference in restriction fragment patterns between adrenal cancer and normal tissues. Cleavage with Hpa II after digestion with Bgl II showed that significantly more DNA was digested into low-molecular weight fragments in adrenal cancer and the normal adrenal gland than those in other normal tissues. By Northern blot analysis, there was difference of signal intensity of hybridizing mRNA between the adrenal cancers and normal adrenal glands. These results suggest that the Eco RI site in the flanking region of the 21-hydroxylase gene may be modified in adrenal cancer tissue, and that inadequate 21-hydroxylase is present in some forms of adrenal cancers.