Genes for the alpha and beta subunits of phycocyanin.

Phycocyanin (PC) is a light-harvesting protein common to blue-green and red algae. We have isolated the genes for the two apoprotein subunits, alpha and beta, of PC from the blue-green alga Agmenellum quadruplicatum PR-6. We synthesized eight sense-strand tetradecameric oligonucleotide probes that could encode a particular pentapeptide in PC alpha from A. quadruplicatum. Only one probe hybridized with total RNA from this organism. This oligonucleotide showed homology to a unique restriction fragment when used to probe Southern blots of A. quadruplicatum DNA. The probe-homologous 3.1-kilobase pair HindIII fragment was cloned. The nucleotide sequence of a 1.7-kilobase pair segment of this clone was determined. Two open reading frames are contained in this region, which correspond in deduced amino acid sequence to PC alpha and PC beta subunits. Both coding sequences are in the same orientation, separated by 105 base pairs, with the PC beta gene 5' to the PC alpha gene. Each gene has a Shine-Dalgarno-type sequence near the initiation codon. Codon frequencies in the two genes may be correlated with the abundance of their products. The deduced amino acid sequences of the gene products show considerable homology with the alpha and beta PC subunits from other species.     

Tandem duplication within a type II collagen gene (COL2A1) exon in an individual with spondyloepiphyseal dysplasia.

We have characterized a mutation in the type II collagen gene (COL2A1) that produces a form of spondyloepiphyseal dysplasia. The mutation is an internal tandem duplication of 45 base pairs within exon 48 and results in the addition of 15 amino acids to the triple-helical domain of the alpha 1 chains of type II collagen derived from the abnormal allele. Although the repeating (Gly-Xaa-Yaa)n motif that characterizes the triple-helical domain is preserved, type II collagen derived from cartilage of the affected individual contains a population with excessive posttranslational modification, consistent with a disruption in triple-helix structure. The mutation is not carried by either parent, indicating that the phenotype in the affected individual is due to a new dominant mutation. DNA sequence homology in the area of the duplication suggests that the mutation may have arisen by unequal crossover between related sequences, a proposed mechanism in the evolution and diversification of the collagen gene family.     

Isolation and characterization of overlapping genomic clones covering the chicken alpha 2 (type I) collagen gene.

A series of overlapping recombinant clones, which cover the alpha 2 (type I) collagen gene, have been isolated by stepwise screening of two libraries of chicken genomic DNA fragments. The first genomic clone was isolated by using a cloned cDNA containing alpha 2 collagen DNA sequences as hybridization probe. The other clones were obtained by a sequence of screenings using defined fragments of the successive genomic clones as hybridization probes. Several types of experiments indicated that the DNA of these clones are truly overlapping and span 55 kilobase pairs of contiguous DNA sequences in the chicken genome. Sequence analysis of small DNA segments of some of these clones confirm that they contain coding sequences which specify alpha 2 collagen. Electron microscopic analysis of hybrids between type I alpha 2 collagen mRNA and the overlapping genomic clones indicates that the chicken alpha 2 collagen gene has a length of at least 37 kilobases, about 7.4 times longer than the corresponding translatable cytoplasmic mRNA. The coding information for alpha 2 collagen is distributed in more than 50 coding sequences which are interrupted by intervening sequences of various sizes. The structure of the gene implies that the conversion of precursor RNA to mature mRNA for alpha 2 collagen includes at least 50 splicing events.     

Exon/intron organization of the chicken type II procollagen gene: intron size distribution suggests a minimal intron size.

Overlapping genomic clones have been isolated that contain the alpha chain and COOH-terminal propeptide coding regions of the chicken type II procollagen gene. All type II procollagen exon sequences present in these clones have been identified and mapped by DNA sequencing. These include 43 exons coding for the alpha-chain triple helix, 1 exon coding for the junction between the COOH-terminal propeptide and the alpha-chain region, and 3 exons coding for the COOH-terminal propeptide and 3' noncoding sequences. With the exception of one additional intron between 2 exons coding for amino acids 568-585 and 586-603, exon-intron boundaries have been conserved when compared with genes for all other characterized genes for fibrillar collagens. The chicken type II procollagen gene differs from most other collagen genes in having introns of considerably smaller average size. The size distribution of the introns suggests that approximately equal to 80 base pairs may be a minimal functional size for introns in this gene. This size of intron may be necessary in a gene with a very large number of small exons to prevent aberrant splicing from removing exon sequence together with intron sequence.     

Large introns in the 3'' end of the gene for the pro alpha 1 (IV) chain of human basement membrane collagen.

Using a recently characterized cDNA clone (HT-21) coding for the pro alpha 1 (IV) chain of human type IV procollagen, we have isolated three clones from a bacterio-phage lambda Charon 4A library of human genomic DNA. The intron/exon structure of the pro alpha 1 (IV) genomic clones was analyzed by heteroduplex electron microscopy and nucleotide sequencing. The analysis showed that the introns separating exons 2-9 are large and have a total length of over 12,000 base pairs (bp). Six of seven exons at the 3' end of the gene coded for -Gly-Xaa-Yaa-repeats of the collagenous part of the chain. Five of the -Gly-Xaa-Yaa- coding exons (numbers 5-9) varied in size between 72 bp and 134 bp, and none of them were 54 bp or multiples thereof. A sixth exon (exon 4) was a junction exon containing 71 bp coding for -Gly-Xaa-Yaa- sequences and 142 bp coding for the carboxyl-terminal noncollagenous domain (NC-1). The seventh exon (exon 3, 178 bp) coded for sequences of the NC-1 domain. Five of the six -Gly-Xaa-Yaa- coding exons began with the second base coding for glycine, and only one exon began with a complete glycine codon at the 5' end. The results (i) suggest that the gene for the pro alpha 1(IV) chain of human basement membrane collagen is significantly larger than the genes for fibrillar collagens and (ii) show that it lacks the 54-bp exon repeats characteristic of fibrillar collagen genes.     

Products of two common alleles at the locus for human placental alkaline phosphatase differ by seven amino acids.

Amino-terminal amino acid sequences (42 residues) were determined for the products of the three common alleles at the human placental alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] gene locus. The sequences differ at position 3, which is proline in types 1 and 2 but is leucine in type 3. cDNA libraries were constructed in phage lambda gt11 and used to isolate clones covering the coding regions of types 1 and 3 cDNAs. Comparison of the deduced amino acid sequences of the types 1 and 3 proteins showed 7 differences out of 513 amino acids, each due to a single base substitution. cDNA sequence comparisons showed three silent substitutions in the coding regions and three base differences in the greater than 1 kilobase pairs of 3' untranslated sequences.     

Spontaneous changes in nucleotide sequence in proviruses of spleen necrosis virus, an avian retrovirus.

We determined the nucleotide sequence of about 1 kilobase of DNA 3' to the 5' long terminal repeat of three noninfectious ad one infectious proviral DNA clones of spleen necrosis virus, an avian retrovirus, to determine if the types of nucleic acid changes involved in retrovirus mutation shed light on special features of retrovirus replication. An open reading frame was found starting 411 base pairs from the end of the long terminal repeat. It contained sequences coding for the 36 amino acids at the amino terminus of the p30 of a related reticuloendotheliosis virus [Oroszlan, S., Barbacid, M., Copeland T., Aaronson, S. A. & Gilden, R. V. (1981) J. Virol. 39, 845-854]. Therefore, the open reading frame represents the 5' end of the gag gene. A mutation in one noninfectious provirus changed the initiation codon for the gag polypeptide; a mutation in another noninfectious provirus caused premature termination of gag polypeptide synthesis; and a nontandem duplication into gag resulting from a mistake in initial (+) strand DNA synthesis changed amino acids and the reading frame in a third noninfectious provirus. These mutations appear to be responsible for the lack of infectivity of these provirus clones and indicate a higher relative frequency of mutation in this region of the genome. In addition, all four clones have multiple other mutations. These mutations are mostly base pair substitutions and many are clustered for any one clone, reflecting certain special features of retrovirus replication.     

Identification of genes for the constant region of rabbit T-cell receptor beta chains.

We describe cDNA clones from thymus mRNA of a young rabbit that have sequences highly homologous to the human and murine T-cell receptor beta-chain constant region (C beta). In rabbit, man, and mouse there is a conserved extra cysteine in the constant region that could lead to a free thiol group or alternative disulfide bond formation depending on the locations and total numbers of cysteines in assembled receptor molecules. A cDNA clone (CL ANA 11) with 571 bases 5' of the C beta coding sequence has an open reading frame starting at a methionine codon that encodes 141 amino acids in frame with the C beta sequence. The encoded sequence has no resemblance to known immunoglobulin or beta-chain variable regions or other known proteins. An oligonucleotide probe from the 5' end of the encoded protein hybridizes to an approximately equal to 2-kilobase genomic DNA fragment that contains C beta gene sequences and to an approximately equal to 8-kilobase mRNA species in the thymus mRNA preparation from which the clone was derived. Within the 5' coding sequence there is a stretch of 211 bases containing strings of alternating purines and pyrimidines that may form Z-DNA. The sequence of the last 55 base pairs adjacent to C beta resembles the corresponding segment of mouse cDNA clone 86T3 that contains sequence 5' of the mouse C beta 1 gene. Although the function of a potential protein encoded by the 5' end of CL ANA 11 is unknown, it could play a role in regulation of thymocyte growth and differentiation.     

The ovalbumin gene: Cloning of the natural gene

The structural ovalbumin DNA sequences are not contiguous and are separated by multiple "intervening regions" in native chicken DNA. EcoRI, a restriction endonuclease that does not cleave the structural ovalbumin DNA sequences, digests the natural ovalbumin gene into three distinct fragments of 2.4, 1.8, and 9.5 kilobase pairs in length by cleaving within these "intervening regions." The 2.4-kilobase pair fragment contains only about 450 nucleotide pairs of coding sequence, with the rest being intervening sequences. This DNA fragment was cloned in bacteria by using the certified EK2 vector lambdagtWES.lambdaB after enrichment from total EcoRI-digested chicken DNA by a combination of RPC-5 column chromatography and preparative agarose gel electrophoresis. Five out of approximately 20,000 recombinant phage plaques were capable of hybridizing with a (32)P-labeled Hha I fragment of a recombinant plasmid pOV230 containing the entire structural ovalbumin gene. DNA amplified in these recombinant phages, lambdagtWES.OV2.4, was shown to contain the same restriction endonuclease cleavage sites as in the 2.4-kilobase pair EcoRI fragment previously determined by restriction mapping of total genomic chicken DNA. The intervening sequences were allowed to hybridize with excess total chicken DNA and oviduct nuclear RNA after nick-translation. They were found to be unique chicken DNA sequences, and appeared to be transcribed in their entireties during gene expression. Like the structural gene sequences, the expression of the intervening sequences is also inducible by steroid hormones.     

Non-immunoglobulin-associated DNA rearrangements in mouse plasmacytomas.

We have characterized a class of DNA rearrangements in plasmacytomas. These recombination events involve a DNA sequence whose origin is outside of the locus of the heavy chain constant region genes, CH. Therefore, we choose to refer to this sequence as non-immunoglobulin-associated rearranging DNA (NIARD). We have isolated two abortively rearranged C alpha genes, generated by NIARD events from the alpha-producing J558 myeloma. Restriction endonuclease maps of these sequences reveal two possible recombination sites in NIARD that are separated by approximately 6.5 kilobase pairs of DNA (defined as 5' and 3' sites). A NIARD rearrangement occurs in 15 out of 20 plasmacytomas tested, including gamma 3-, gamma 1-, gamma 2b-, gamma 2a-, and alpha-producers, but this event usually does not involve a CH switch (S) region. In fact, only S alpha appears to accept NIARD. However, NIARD did not undergo a rearrangement in eight IgA-producing hybridomas tested. One germ-line copy of NIARD (a 22-kilobase pair EcoRI fragment) is retained in all plasmacytomas. NIARD does not appear to possess repetitive DNA sequences homologous to S mu or S alpha. We discuss the possible role and implications of NIARD-like sequence rearrangements in allelic exclusion and chromosomal translocation events in plasmacytomas.     

Cloning and sequence of cDNA coding for alpha 1-antitrypsin.

Recombinant plasmids containing human and baboon cDNA have been screened for alpha 1-antitrypsin, a major serine protease inhibitor present in blood. One plasmid, designated pBa alpha 1a2, was found to contain a cDNA insert of 1352 base pairs coding for the baboon inhibitor. It included 45 nucleotides that code for 15 amino acids present in the amino-terminal signal sequence of the protein, 1182 nucleotides that code for 394 amino acids in the mature protein, a stop codon, and a noncoding region of 76 nucleotides. Comparison of the amino acid sequences of baboon alpha 1-antitrypsin, human antithrombin III, and chicken ovalbumin indicated that these three proteins are about 230% homologous. A second plasmid, designated pH alpha 1a1, was found to contain a human cDNA insert of 306 base pairs. This plasmid coded for 69 amino acids present in the carboxyl-terminal region of human alpha 1-antitrypsin. The human and baboon cDNAs and their amino acid sequences are greater than 96% homologous.     

Isolation and analysis of genomic DNA clones encoding the third component of mouse complement.

A gene library was constructed with DNA from strain A mice by using the phage lambda vector lambda 1059. By screening with cloned cDNA for the third component of mouse complement, C3, four different C3 genomic clones were isolated from this library. Two of the recombinant phages carry insertions of 14 and 18 kilobase pairs, respectively, which together cover one complete copy of the C3 gene and several hundred nucleotides of its 5' and 3' flanking sequences. The distance from the 5' end of the gene, which includes the hexanucleotide T-A-T-A-A-A and a translation initiation codon, to its 3' end as defined by the poly(A) attachment site is 24 kilobase pairs. From the genomic DNA sequence, a signal peptide of 24 amino acid residues is predicted at the NH2 terminus of the initial translation product. The signal peptide and the next two amino acids are encoded by the first exon of this gene.     

Expression and novel structure of a collagen gene in Drosophila.

We report the structure and developmental expression of collagen gene sequences in Drosophila melanogaster. Collagen-like genomic clones were isolated by screening a Drosophila genomic library with a chicken pro alpha 2(I) cDNA clone as a hybridization probe. A 1.5-kilobase (kb) DNA sequence from a 9.2-kb DNA clone (pDCg1) is presented. Unlike the highly fragmented genes for vertebrate type I collagen, there is no evidence of a 54-base-pair primordial unit within this gene segment. Instead, the fragment is composed of two large coding sequences. Together they specify a sequence of 469 amino acids. This collagen product is composed almost entirely of the Gly-X-Y repeat characteristic of peptides involved in triple helix formation. Within the polypeptide there are four minor discontinuities in the Gly-X-Y pattern. Similar interruptions have been observed in a mouse basement membrane collagen protein sequence. Therefore, the Drosophila collagen gene may encode a nonfibrous collagen such as a basement membrane or cuticle collagen or a novel collagenous protein. By using the DNA segment of known sequence as a hybridization probe, a developmental sequence of polyadenylylated RNA samples was screened for the presence of homologous sequences. A RNA species 6.4 kb in length was detected as a prominent band only in the first- and second-instar larval stages. This pattern of developmental hybridization correlates with the production of the cuticle and basement membranes, and the large size of the RNA is consistent with its identification as a collagen-encoding RNA.     

Characterization of an intronless collagen gene family in the marine sponge Microciona prolifera.

Two independent clones from the genomic DNA of a marine sponge Microciona prolifera were isolated by hybridization to the Caenorhabditis elegans Col-1 gene and one clone was obtained from genomic DNA by PCR. They contain open reading frames (MpCol1, MpCol2, MpCol3, MpCol4) capable of coding for a family of collagens different from those previously found in sponges. Southern blotting of genomic DNA suggested the presence of several other homologous genes. cDNA clones covering most of the triple-helical coding domain and the 3' untranslated region of MpCol1 were isolated by specific primers and reverse PCR. Two cDNA clones end in the middle of an AATAAA sequence 170 bp downstream from the translation stop codon of MpCol1. The putative NH2-terminal noncollagenous peptide is composed of only seven amino acid residues. The 1074-bp triple-helical coding region is not interrupted by intervening sequences. It codes for a polypeptide of 120 Gly-Xaa-Yaa triplets with only one short interruption near the COOH terminus. A putative N-glycosylation sequence (Asn-Gly-Ser), three Arg-Gly-Asp triplets known as cell recognition peptides, frequent Lys residues in the Yaa position (which are templates for hydroxylation), several Lys-Gly-Asn/Xaa-Arg peptides known as the lysyl oxidase recognition site, and long stretches without imino acids could be found within the triple-helical domain. The short COOH-terminal noncollagenous domain closely resembles that of nematode cuticular collagens and vertebrate nonfibrillar collagens. Our results strongly support the idea that the diversity of collagen genes and gene families found in higher organisms already existed in sponge.     

Nucleotide sequence of the mRNA encoding the pre-alpha-subunit of mouse thyrotropin.

We have constructed and cloned in bacteria recombinant DNA molecules containing DNA sequences coding for the precursor of the alpha subunit of thyrotropin (pre-TSH-alpha). Double-stranded DNA complementary to total poly(A)+RNA derived from a mouse pituitary thyrotropic tumor was prepared enzymatically, inserted into the Pst I site of the plasmid pBR322 by using poly(dC).poly(dG) homopolymeric extensions, and cloned in Escherichia coli chi 1776. Cloned cDNAs encoding pre-TSH-alpha were identified by their hybridization to pre-TSH-alpha mRNA as determined by cell-free translations of hybrid-selected and hybrid-arrested RNA. The nucleotide sequences of two cDNAs (510 and 480 base pairs) were determined with chemical methods and corresponded to much of the region coding for the alpha subunit and the 3' untranslated region of pre-TSH-alpha mRNA. The sequence of the 5' end of the mRNA was determined from cDNA synthesized by using total mRNA as template and a restriction enzyme DNA fragment as primer. Together these sequences represented greater than 90% of the coding and noncoding regions of full-length pre-TSH-alpha mRNA, which was determined to be 800 bases long. The amino acid sequence of the pre-TSH-alpha deduced from the nucleotide sequence showed a NH2-terminal leader sequence of 24 amino acids followed by the 96-amino-acid sequence of the apoprotein of TSH-alpha. There is greater than 90% homology in the amino acid sequences among the murine, ruminant, and porcine alpha subunits and 75-80% homology among the murine, equine, and human alpha subunits. Several regions of the sequence remain absolutely conserved among all species, suggesting that these particular regions are essential for the biological function of the subunit. The successful cloning of the alpha subunit of TSH will permit further studies of the organization of the genes coding for the glycoprotein hormone subunits and the regulation of their expression.