Two SP-C genes encoding human pulmonary surfactant proteolipid

Human pulmonary surfactant proteolipid of Mr = 5,000, now termed surfactant protein C (SP-C), is produced by proteolytic processing of an Mr = 22,000 precursor. The active hydrophobic peptide imparts surface active properties to pulmonary surfactant phospholipids. We have determined the entire nucleotide sequence of two distinct genes encoding SP-C from a genomic library prepared from human leukocytes. SP-C genes were encoded by approximately 3.0 kilobase pairs of DNA containing six exons and five introns. In both genes, the active hydrophobic region of the polypeptide was located in the second exon that encodes a peptide of 53 amino acids. The entire nucleotide sequences of the two classes of SP-C genes differed by only 1%. Two cDNAs encoding SP-C were distinguished on the basis of an 18-nucleotide deletion at the beginning of the fifth exon; no such deletion was detected within the two classes of SP-C genes. Comparison of the 3'-untranslated regions of SP-C cDNA clones and the two classes of genomic clones demonstrated that cDNAs with and without the 18-base pair deletion could be derived from both of the genes. This 18-base pair deletion occurs in nucleotide sequences compatible with two distinct RNA splice sites. One additional cDNA clone showed the addition of an 8-base pair insert at the end of exon 5, which was also compatible with two distinct splice sites. Both classes of SP-C genes were represented by cDNAs, demonstrating that both classes of genes are actively transcribed. The two SP-C genes were readily distinguished on the basis of their nucleotide sequences and restriction fragment analyses of their flanking DNA. Two distinct classes of human SP-C genes are transcribed, and the heterogeneity in the SP-C RNAs appears to result from differential splicing.     

Phenotype-associated env gene variation among eight related human immunodeficiency virus type 1 clones: evidence for in vivo recombination and determinants of cytotropism outside the V3 domain.

The nucleotide sequences of the env genes of eight phenotypically heterogeneous human immunodeficiency virus type 1 (HIV-1) clones recovered from a single individual within a 3-week period were compared. In addition, the accessory gene sequences for four of these clones were obtained. Variation among most accessory genes was limited. In contrast, pronounced phenotype-associated sequence variation was observed in the env gene. At least three of these clones most likely resulted from genetic recombination events in vivo, indicating that this phenomenon may account for the emergence of proviruses with novel phenotypic properties. Within the env genes of the eight clones, four domains could be defined, the sequence of each of which clustered in two groups with high internal homology but 11 to 30% cluster variation. The extensive env gene variation among these eight clones could largely be explained by the unique manner in which the alleles of these four domains were combined in each clone. Experiments with chimeric proviruses demonstrated that the HIV-1 env gene determined the capacity to induce syncytia and tropism for T-cell lines. Amino acids previously shown to be involved in gp120-CD4 and gp120-gp41 interaction were completely conserved among these eight clones. The finding of identical V3 sequences in clones differing in tropism for primary monocytes and T-cell lines demonstrated the existence of determinants of tropism outside the env V3 region.     

Isolation and characterization of human VkIII germ-line genes. Implications for the molecular basis of human VkIII light chain diversity

To understand the relative importance of germ-line genes in the generation of the functional human antibody repertoire, it is first necessary to define the number of variable region genes and to determine their fine structure. We have focused on the human VkIII variable region gene family because of its association with autoantibodies. A human genomic library was screened with a VkIII cDNA probe and subsequently with a VkIII germ-line gene probe. Seven different VkIII clones were isolated and characterized by restriction mapping and sequence analyses. Three clones have identical restriction enzyme sites over a 12-kilobase (kb) region, contain identical sequences over an 895-base pair (bp) region, and thus are likely to be different isolates of the same human VkIII gene. Another two clones have identical restriction enzyme sites over a 5-kb region, are identical over a stretch of 905 bp sequenced, and likely represent independent isolates of another human VkIII gene. The remaining two VkIII clones consist of two additional VkIII genes which are homologous to each other, but are quite different from the first two VkIII genes. Thus, four new human VkIII genes were defined. Together with four other VkIII genes previously isolated by other investigators, a total of eight human VkIII germ-like genes have now been described. A comparison of the deduced amino acid sequences of these genes with the reported amino acid sequences of all human VkIII light chains suggests that at least one additional VkIII gene exists in the germ line. Among the eight identified human germ-line VkIII genes, three are pseudogenes. Of the remaining five potential functional genes, one gene seems to encode a majority of the VkIII light chains which have been sequenced. Possible explanations for this phenomenon are discussed.     

Isolation of cloned DNA sequences containing ribosomal protein genes from Saccharomyces cerevisiae.

Yeast mRNA enriched for ribosomal protein mRNA was obtained by isolating poly(A)+ small mRNA from small polysomes. A comparison of cell-free translation of this small mRNA and total mRNA, and electrophoresis of the products on two-dimensional gels which resolve most yeast ribosomal proteins, demonstrated that a 5-10 fold enrichment for ribosomal protein mRNA was obtained. One hundred different recombinant DNA molecules possibly containing ribosomal protein genes were selected by differential colony hybridization of this enriched mRNA and unfractionated mRNA to a bank of yeast pMB9 hybrid plasmids. After screening twenty-five of these candidates, five different clones were found which contain yeast ribosomal protein gene sequences. The yeast mRNAs complementary to these five plasmids code for 35S-methionine-labeled polypeptides which co-migrate on two-dimensional gels with yeast ribosomal proteins. Consistent with previous studies on ribosomal protein mRNAs, the amounts of mRNA complementary to three of these cloned genes are controlled by the RNA2 locus. Although two of the five clones contain more than one yeast gene, none contain more than one identifiable ribosomal protein gene. Thus there is no evidence for "tight" linkage of yeast ribosomal protein genes. Two of the cloned ribosomal protein genes are single-copy genes, whereas two other cloned sequences contain two different copies of the same ribosomal protein gene. The fifth plasmid contains sequences which are repeated in the yeast genome, but it is not known whether any or all of the ribosomal protein gene on this clone contains repetitive DNA.     

Linkage arrangement of human placental lactogen and growth hormone genes

Human placental lactogen (hPL) and growth hormone (hGH) are two hormones thought to have evolved from a common ancestral gene (along with prolactin), yet they have quite different functions and specificities. The nucleic acid sequences of the respective cDNAs of the two genes share considerable homology, as well as the existence of multiple forms of each gene within the genome. In this study we report on the linkage arrangement of several genes from this group. Two hPL-like genes as well as an hGH gene are shown to be linked within a 38-kilobase pair region of DNA. Linkage between a variant hGH gene and an hPL gene is also shown. The orientation and structural organization of these genes was previously established using 5'- and 3'-specific probes from a placental lactogen cDNA clone and detailed restriction endonuclease mapping. Restriction fragments from the overlapping clones were verified by comparison to digests of high molecular weight genomic DNA. In addition, the location of a specific class of repetitive DNA sequences, the Alu family, was mapped on these clones using the recombinant clone BLUR 8. All members of this multigene family have Alu repeat sequences either immediately flanking their 3' or 5' untranslated regions or within their intervening sequences.     

Isolation of Expressed Sequences Encoded by the Human Xq Terminal Portion Using Microclone Probes Generated by Laser Microdissection

The genes that cause a variety of neurologic and neuromuscular disorders have been mapped to the distal region of Xq. In an effort to isolate genes from this area, a regional genomic library of the distal 30% of Xq was constructed from a single metaphase spread by means of laser microdissection and single unique primer-polymerase chain reaction. Using pooled probes of 1000 clones from the genomic library, human brain cDNA libraries were screened for expressed sequences encoded by this region. From the 250,000 cDNA clones screened so far, 10 nonoverlapping sequences that mapped back to the target portion were isolated. The complete nucleotide sequences of these cDNA clones have been determined. Analysis of the sequences indicates that none has significant similarity to previously characterized primate genes. One sequence mapping to Xq27.3-qter contained an open reading frame of 281 amino acids and was expressed in every tissue tested. This gene, as well as others isolated in this manner, may prove to be a candidate gene for heritable disorders mapping to this region.     

Embryonic simple epithelial keratins 8 and 18: chromosomal location emphasizes difference from other keratin pairs.

The keratins 8 and 18 of simple epithelia differ from stratified epithelial keratins in tissue expression and regulation. To examine the specific properties of human keratin 8, we cloned and sequenced the cDNA from a placental mRNA expression library and defined the optimum state of such clones for expression in bacterial plasmid vectors. Using the polymerase chain reaction we identified and sequenced three introns and located the single active gene for keratin 8, out of a background of 9 to 24 pseudogenes, on chromosome 12. This chromosome contains several genes for type II keratins and also the gene for keratin 18, the type I keratin that is coexpressed with keratin 8. This location of both members of a keratin pair on a single chromosome is thus far unique among the keratin genes; it is consistent with the hypothesis that keratins 8 and 18 may be closer to an ancestral keratin gene than the keratins of more highly differentiated epithelia.     

Multiple cDNA sequences and the evolution of bovine stomach lysozyme

To investigate the origin of stomach expression of lysozyme in ruminants; we surveyed clones from a cow stomach cDNA library with a lysozyme cDNA probe. Ten percent of the clones in this library were lysozyme-specific. Thirty of the lysozyme clones were sequenced, and seven types of lysozyme mRNA sequence were found. They encode the three previously identified stomach isozymes of lysozyme. The seven sequences are closely related to one another and represent the products of a minimum of 4 of the approximately 10 cow lysozyme genes detected by genomic blotting. The most abundant form of stomach lysozyme (form 2) is encoded by at least two genes, whereas forms 1 and 3 are possibly each encoded by only one gene. The number of genes encoding each isozyme appears to contribute the largest factor in the relative abundance of each isozyme. The multiple lysozyme genes expressed in the cow stomach are the result of gene duplications that occurred during ruminant evolution. The recruitment of lysozyme as a major enzyme in the stomach may thus have involved an early regulatory event and a later 4-7-fold increase in expression allowed by gene amplification. During this period, the amino acid sequences of these lysozymes have been evolving more slowly than those of nonruminant lysozymes.     

The phylogenetic affiliation of an uncultured population of ammonia-oxidizing bacteria harboring environmental sequences of amoA cluster-3

We investigated the phylogenetic diversity of ammonia-oxidizing bacteria (AOB) in Yellow Sea continental shelf sediment by the cloning and sequencing of PCR-amplified amoA and 16S rRNA genes. Phylogenetic analysis of the amoA-related clones revealed that the diversity of AOB was extremely low at the study site. The majority (92.7%) of amoA clones obtained belonged to a single cluster, environmental amoA cluster-3, the taxonomic position of which was previously unknown. Phylogenetic analysis on AOB-specific 16S rRNA gene sequences also demonstrated a very low diversity. All of the cloned 16S rRNA gene sequences comprised a single phylotype that belonged to the members of uncultured Nitrosospira cluster-1, suggesting that AOB belonging to the uncultured Nitrosospira cluster- 1 could carry amoA sequences of environmental amoA cluster-3.     

Three types of rat U1 small nuclear RNA genes with different flanking sequences are induced to express in vivo

There are about 50 copies of U1 RNA genes/pseudogenes in the rat genome. To date, we have isolated so far 25 phage clones carrying a U1 RNA gene/pseudogene from two rat genomic libraries. The 12 clones were selected by hybridization with the U1 RNA coding region under a stringent condition, and were mapped and sequenced. Here, we report three types of U1 RNA genes with different flanking sequences, all of which were shown to be induced to express in vivo by transfection with their polylinker-inserted maxi U1 RNA genes into cultured rat cells. Although these three classes of U1 RNA genes have few homologous flanking sequences, they provide both upstream and downstream of the genes two conserved blocks, which may possibly play an important role in U1 RNA expression.     

The ornithine aminotransferase-encoding gene family of rat: cloning, characterization, and evolutionary relationships between a single expressed gene and three pseudogenes

As a first step towards understanding the molecular mechanisms through which the expression of the gene (OAT) encoding ornithine aminotransferase (OAT) is regulated in a tissue-specific manner, we have used a near full length OAT cDNA to isolate related sequences from a rat genomic DNA library. Twenty-one unique clones representing five contigs and spanning approximately 140 kb of genomic DNA were isolated and characterized. From these clones we have identified a single expressed OAT gene and three processed pseudogenes. The comparison of the EcoRI, BamHI, and HindIII fragments contained within these genomic clones with those detected in total genomic DNA by the cDNA probe suggests that essentially all of the OAT-related sequences in the rat genome have been isolated. Thus, the tissue-specific regulation of OAT gene expression appears to be effected through a single expressed gene. Data are presented which suggest that the OAT-1, OAT-2, and OAT-3 pseudogenes arose approximately 28.5, 7.3, and 25.1 Myr ago, respectively. Mutation rates are presented for each codon position of the expressed rat and human OAT genes. The region of the rat genome flanking the boundary of the OAT-3 pseudogene is of additional interest as it shares considerable identity to sequences contained within expressed genes and flanking other processed pseudogenes.     

CpG island libraries from human Chromosomes 18 and 22: landmarks for novel genes

CpG islands are found at the 5′ end of approximately 60% of human genes and so are important genomic landmarks. They are concentrated in early-replicating, highly acetylated gene-rich regions. With respect to CpG island content, human Chrs 18 and 22 are very different from each other: Chr 18 appears to be CpG island poor, whereas Chr 22 appears to be CpG island rich. We have constructed and validated CpG island libraries from flow-sorted Chrs 18 and 22 and used these to estimate the difference in number of CpG islands found on these two chromosomes. These libraries contain normalized collections of sequences from the 5′ end of genes. Clones from the libraries were sequenced and compared with the sequence databases; one third matched ESTs, thus anchoring these ESTs at the 5′ end of their gene. However, it was striking that many clones either had no match or matched only existing CpG island clones. This suggests that a significant proportion of 5′ gene sequences are absent from databases, presumably either because they are difficult to clone or the gene is poorly expressed and/or has a restricted expression pattern. This point should be taken into consideration if the currently available libraries are those used for the elucidation of complete, as opposed to partial, gene sequences. The Chr 18 and 22 CpG island libraries are a sequence resource for the isolation of such 5′ gene sequences from specific human chromosomes. Received: 15 November 1999 / Accepted: 31 January 2000     

Internal organization of the major adult alpha- and beta-globin genes of X. laevis

We describe the isolation of two recombinant lambda phages, each containing genomic DNA fragments encoding both the major adult alpha- and beta-globin mRNAs of X. laevis. The DNA fragment in the two clones have restriction maps which indicate that they are each derived from a different member of the pair of alleles present in the heterozygote used as the source of DNA for cloning. The characterization of these two clones by restriction mapping, R looping and DNA sequencing shows that the alpha 1- and beta 1-globin genes lie in the orientation separated by 7.7 kb of DNA. There are two introns in the alpha 1-globin gene and two in the beta 1-globin gene, and they interrupt the genes at exactly the same positions as the introns found in all known mammalian alpha- and beta-globin genes. The exon sequences proximal to the introns show a much higher degree of homology with mammalian sequences than the sequences distal to intron/exon junctions, and the introns in the beta 1-globin gene of X. laevis are very similar in length to the corresponding introns in the beta-globin genes of several mammals and the chicken.