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PubMed2026

Advances in CRISPR Base Editing: From Molecular Evolution to Therapeutic Applications in Genomic Medicine.

CRISPR-Cas9 systems revolutionized gene editing, but inherent drawbacks, namely DNA double-strand breaks (DSBs) and the difficulty of achieving precise repairs (due to low HDR efficiency), led researchers to invent new, more accurate gene editing tools. Base editing represents a significant leap forward, enabling targeted single-nucleotide conversions directly on the DNA without DSBs or donor templates. The core technology involves fusing catalytically dead or nickase Cas proteins to DNA deaminase enzymes. Cytosine base editors (CBEs) convert C•G to T•A pairs, while adenine base editors (ABEs) change A•T to G•C. These editors exploit the deaminase function within the R-loop structure formed by Cas binding and co-opt endogenous DNA repair mechanisms for precision. While offering improved efficiency and editing precision, base editing faces persistent challenges, such as off-target effects, bystander edits, delivery and ethical concerns. Continuous engineering efforts have refined these tools, enhancing accuracy, expanding targetability and reducing unwanted edits. The base editing arsenal has also broadened to include C-to-G base editors (CGBEs), dual A&C editors and versions targeting organelles. Successful preclinical studies demonstrating the correction of mutations responsible for the disease have paved the way for clinical trials, which are now testing therapies for conditions like sickle cell disease, β-thalassaemia and hypercholesterolemia using various delivery systems. This review explores CRISPR base editing's origins, mechanisms of action, potential therapies and current restrictions, pointing to its broadening impact on medical genetics.

Humans↗

PubMed1987

Organization of rRNA genes in Mycobacterium bovis BCG.

The number of rRNA genes in Mycobacterium bovis BCG was examined by Southern hybridization of end-labeled 5S, 16S, and 23S rRNAs with BamHI, PstI, and SalI digests of M. bovis BCG DNA. Each RNA probe gave only one radioactive band with three kinds of DNA digest. These results suggest that M. bovis BCG chromosomes may carry only a minimum set of rRNA genes. Hybridization of randomly labeled rRNAs with BamHI, PstI, SalI, BglII, and PvuII digests of DNA from the same organism supported these conclusions. The 6.4-kilobase-pair SalI fragment containing the entire structural genes for both 16S and 23S rRNAs was cloned into pBR322. The cloned fragment was characterized by restriction endonuclease mapping, DNA-RNA hybridization analysis, and the R-loop technique. The results indicated that the fragments contained rRNA genes in the following order: 16S, 23S, and 5S rRNA genes. No tRNA gene was detected in the spacer region between the 16S and 23S rRNA genes, but one was found downstream of the 23S rRNA and 5S rRNA genes.

Cloning, Molecular↗

PubMed1979

Molecular cloning of the Harvey sarcoma virus closed circular DNA intermediates: initial structural and biological characterization.

Supercoiled Harvey sarcoma virus (Ha-SV) DNA was extracted from newly infected cells by the Hirt procedure, enriched by preparative agarose gel electrophoresis, and digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized Ha-SV DNA was then inserted into lambda gtWESlambda B at the EcoRI site and cloned in an approved EK2 host. Ha-SV DNA inserts from six independently derived recombinant clones have been analyzed by restriction endonuclease digestion, molecular hybridization, electron microscopy, and infectivity. Four of the Ha-SV DNA inserts were identical, contained about 6.0 kilobase pairs (kbp), and comigrated in agarose gels with the infectious, unintegrated, linear Ha-SV DNA. One insert was approximately 0.65 kbp smaller (5.35 kbp) and one was approximately 0.65 kpb larger (6.65 kpb) than the 6.0 kpb inserts. R-looping with Ha-SV RNA revealed that the small (5.35 kbp) insert contained one copy of the Ha-SV RNA. Preliminary restriction endonuclease digestion of the recombinant DNAs suggested that the middle-size inserts contained a 0.65-kbp tandem duplication of sequences present only one in the small-size insert; this duplication corresponded to the 0.65-kpb terminal duplication of the unintegrated linear Ha-SV DNA. The large-size insert apparently contained a tandem triplication of these terminally located sequences. DNA of all three sized inserts induced foci in NIH 3T3 cells, and focus-forming activity could be rescued from the transformed cells by superinfection with helper virus. Infectivity followed single-hit kinetics, suggesting that the foci were induced by a single molecule.

Animals↗

PubMed1984

Nucleotide sequence of an immediate-early frog virus 3 gene.

We have used "gene walking" with synthetic oligonucleotides and M13 dideoxynucleotide sequencing techniques to obtain the complete coding and flanking sequences of the gene encoding a major immediate-early RNA (molecular weight, 169,000) of frog virus 3. R-loop mapping of the cloned XbaI K fragment of frog virus 3 DNA with immediate-early RNA from infected cells showed that an RNA of approximately 500 to 600 nucleotides (the right size to code for the immediate-early viral 18-kilodalton protein of unknown function) hybridized to a region within 100 base pairs of one end of the XbaI K fragment; no evidence for splicing was observed in the electron microscope or by single-strand nuclease analysis. Further restriction mapping narrowed the location of the gene to the XbaI end of a 2-kilobase-pair XbaI-Bg/II fragment, which was bidirectionally subcloned into the bacteriophage pair mp10 and mp11 for sequencing. Mung bean nuclease mapping was used to identify both the 5' and the 3' ends of the mRNA. The 5' end mapped within an AT-rich region 19 base pairs upstream from two in-phase AUG start codons that were immediately followed by an open reading frame of 157 amino acids. Another AT-rich sequence was found at -29 base pairs from the 5' end of the mRNA start site; this sequence may function as a TATA box. The 3' end of the message displayed considerable microheterogeneity, but clearly terminated within a third AT-rich region 50 to 60 base pairs from the translation stop codon. The eucaryotic polyadenylic acid addition signal (AATAAA) was not present, a finding to be expected since frog virus 3 mRNA is not polyadenylated. Both the single-stranded mp10 clone of the XbaI-Bg/II fragment and a 15-base oligonucleotide complementary to the region flanking the two AUG translation start codons inhibited translation of the immediate-early 18-kilodalton protein in vitro, confirming the identity of the sequenced gene. As the regulatory sequences of this gene did not resemble those of known eucaryotic genes or of the cytoplasmic vaccinia virus, we conclude that frog virus 3 has evolved unique signals for the initiation and termination of transcription.

Base Sequence↗

PubMed1987

Identification and mapping of human papillomavirus type 1 RNA transcripts recovered from plantar warts and infected epithelial cell cultures.

Multiple spliced transcripts of human papillomavirus type 1 were detected by electron microscopic analysis of R-loops formed with total RNA extracted from plantar warts and with poly(A)+ RNA isolated from cultured keratinocytes infected with human papillomavirus type 1. The 5' ends of the RNAs were mapped to sites in the E7 open reading frame (ORF), just upstream of the E6 ORF and in the upstream regulatory region. Species with 5' ends in E7 accounted for over 95% of all transcripts seen. Two polyadenylation sites were used, one at the end of the early (E) region of the viral DNA, the other at the end of the late (L) region. The most abundant species had a short 5' exon of approximately 100 nucleotides spanning the junction of the E7 and E1 ORFs spliced to a 3' exon of 800 nucleotides in the region with overlapping E2 and E4 ORFs; it was polyadenylated at the end of the E region. This species probably encodes the abundant E4 protein found in plantar warts (F. Breitburd, O. Croissant, and G. Orth, Cancer Cells, vol. 5, in press; J. Doorbar, D. Campbell, R. J. A. Grand, and P. H. Gallimore, EMBO J. 5:355-362, 1986). Other transcripts had exons spanning the E6-E7 ORFs, the E4-E5-L2-L1 ORFs, or the L1 ORF. The infrequent L1 transcript, probably the mRNA coding for the major capsid protein, had the same 5' exon in E7 as the abundant mRNA spliced from E1 and E4 ORFs, suggesting genetic regulation via the choice of the alternative polyadenylation sites or mRNA processing.

Base Sequence↗

PubMed1987

Human papillomavirus types 6 and 11 mRNAs from genital condylomata acuminata.

We have identified and mapped a number of RNA species of human papillomavirus types 6 and 11 from condylomata acuminata by the electron microscopic R-loop technique. Each of the early (E)- and late (L)-region open reading frames (ORFs) deduced from the DNA sequences was represented in one or more transcripts. In addition, RNA species that could encode the modulator of DNA replication and the repressor of transcription, functions recently identified in the genetically similar bovine papillomavirus type 1, were also detected. Some ORFs were 5' proximal in one or more transcripts, whereas others were not 5' proximal in any species, suggesting that internal initiation of translation might be required to gain access to these latter ORFs. Virtually all transcripts had their 5' ends located in the E region and were polyadenylated at one of two sites, i.e., at the end of the E region or at the end of the L region. The great majority of the RNAs were derived from the E region of the genome, with one species approximately 50 to 100 times more abundant than the others. For most of the RNAs, the 5' end mapped near nucleotide 700; minor populations had 5' ends near nucleotide 100 or 1200. By correlating our mapping data with the genomic DNA sequences as well as available RNA structures and cDNA sequences of several papillomaviruses, we predict a number of mRNA splice donor and acceptor sites and suggest that the papillomaviruses have sophisticated usage of ORFs through alternative promoters, mRNA splice sites, and polyadenylation sites.

Anus Neoplasms↗

PubMed2025

Developing a disease-specific accessible transcriptional signature as a biomarker for ataxia with oculomotor apraxia type 2.

BACKGROUND: Genetic ataxias are clinically heterogenous neurodegenerative conditions often involving rare or private mutations and it is often difficult to assign pathogenicity to rare gene variants solely based on DNA sequencing. An effective functional assay from an easy-to-obtain biospecimen would aid this assessment and be of high clinical value. SETX encodes a ubiquitous DNA/RNA helicase crucial for resolving R-loops and maintaining genome stability. Loss-of-function mutations cause a recessive disorder, Ataxia with Oculomotor Apraxia Type 2 (AOA2). METHODS: Here we utilize Weighted Gene Co-expression Network Analysis (WGCNA) from patient blood to construct an AOA2-specific transcriptomic signature as a biomarker to evaluate SETX variants in patients clinically suspected of having AOA2. RESULTS: WGCNA from peripheral blood RNA of 11 AOA2 patients from 7 families initially identified a single gene module that was modestly effective in distinguishing individuals with AOA2 from controls (sensitivity 73%, specificity 97%) and was able to robustly differentiate AOA2 patients from those with genetically distinct, yet phenotypically similar, neurological disorders (sensitivity 100%, specificity 100%). An independent derivation of the transcriptional biomarker identified a dual module model that was able to better distinguish individuals with AOA2 from controls (sensitivity 100%, specificity 97%). As validation, we examined a second cohort of 21 patients from 13 families and demonstrate that this dual module transcriptional biomarker could discriminate patients clinically suspected of AOA2 from controls (57%, 95%CI: 34%-78%). Overall, the transcriptional biomarker was able to separate AOA2 subjects (n = 32) from controls (n = 35) with 72% sensitivity and 97% specificity. Notably, this transcriptomic biomarker enabled verification of the first pathogenic SETX mutation found in a non-canonical transcript, expanding the spectrum of mutations that contribute to AOA2. CONCLUSIONS: Our study identified a transcriptional biomarker that was able to differentiate AOA2 from controls and from other related neurological disorders, consequently expanding the spectrum of known pathogenic mutations. This proof-of-concept study illustrates that transcriptional biomarkers may be used to validate variants of uncertain significance in known genetic diseases.

Humans↗

PubMed1981

DNA electron microscopy.

In recent years DNA electron microscopy has become a tool of increasing interest in the fields of molecular genetics and molecular and cell biology. Together with the development of in vitro recombination and DNA cloning, new electron microscope techniques have been developed with the aim of studying the structural and functional organization of genetic material. The most important methods are based on nucleic acid hybridizations: DNA-DNA hybridization (heteroduplex, D-loop), RNA-DNA hybridization (R-loop), or combinations of both (R-hybrid). They allow both qualitative and quantitative analysis of gene organization, position and extension of homology regions, and characterization of transcription. The reproducibility and resolution of these methods make it possible to map a specific DNA region within 50 to 100 nucleotides. Therefore they have become a prerequisite for determining regions of interest for subsequent nucleotide sequencing. Special methods have been developed also for the analysis of protein-DNA interaction: e.g., direct visualization of specific protein-DNA complexes (enzymes, regulatory proteins), and analysis of structures with higher complexity (chromatin, transcription complexes).

Adsorption↗

PubMed1992

The start site of the Acanthamoeba castellanii ribosomal RNA transcription unit.

The 39S ribosomal RNA (rRNA) precursor has been isolated from Acanthamoeba castellanii. In vitro capping of the isolated RNA verified that it is the primary transcript and identified the 5' nucleotide as pppA. The position of the 5' coding nucleotide on the rRNA repeat unit sequence was identified using Northern blot, R-loop, and S1 nuclease mapping techniques. Dinucleotide priming of an in vitro transcription system stalled because of low initiating nucleotide concentration revealed that ApA maximally stimulates initiation of transcription. All of these results show that the underlined A in the sequence 5'-TATATATAAAGGGAC (RNA-like strand) coincides with the 5' nucleotide of the primary transcript. This identification is compatible with in vitro transcription experiments mapping the promoter for this transcription unit. The initiation sequences of rRNA genes from 14 species are compared, and a weak consensus for the initiator derived: [Formula; see text].

Acanthamoeba↗

PubMed1989

Structural organization of the gene for the alpha 1 chain of human type IV collagen.

The complete exon size and distribution pattern in the gene for the alpha 1 chain of human type IV collagen was determined. Clones covering 145 kilobases (kb) of genomic DNA including 100 kb of the gene itself as well as 25 kb upstream and 20 kb downstream of the gene sequences, respectively, were isolated from lambda phage and cosmid libraries. The overall gene structure was determined by endonuclease restriction mapping and R-loop analyses and all exon sizes by nucleotide sequencing. The characterized clones contained all the coding sequences except for exon 2 whose sequence was determined after its amplification by the polymerase chain reaction. There were four gaps in the intron sequences; the exact size of the gene is unknown. The entire gene is at least 100 kb in size and contains 52 exons whose size distribution is completely different from that of the genes for fibrillar collagens. In the -Gly-X-Y- coding region there are three exons of 99, 90, and 45 base pairs (bp) each and two exons of 27, 36, 42, 51, 54, 63, and 84 bp each. The rest of the exons have sizes between 71 and 192 bp in the collagenous region. About one-half of the -Gly-X-Y- repeat coding exons start with the second base for the codon of glycine, whereas the other half starts (with two exceptions) with a complete glycine codon. The distribution of split versus unsplit codons is uneven in that the first 19 exons of the gene start with a complete codon. The gene contains repetitive sequences in several regions. A 185-nucleotide segment containing 40 copies of CCT flanked by poly(C) and poly(T) sequences was shown to be located adjacent to an exon. The gene has previously been shown to be located head-to-head to the alpha 2(IV) collagen gene at the distal end of the long arm of chromosome 13, such that the first exons of the two genes are separated by as little as 42 bp (Pöschl, E., Pollner, R., and Kühn, K. (1988) EMBOJ. 7,2687-2695; Soininen, R., Huotari, M., Hostikka, S. L., Prockop, D. J., and Tryggvason, K. (1988) J. Biol. Chem. 263, 17217-17220). The results demonstrate that the human alpha 1(IV) collagen gene has a structure distinctly different from the genes for fibrillar collagens and also that it is considerably larger than any collagen gene characterized to date.

Amino Acid Sequence↗

PubMed1985

Structure of the promoter of the rat type II procollagen gene.

We have isolated several overlapping genomic clones which contain the 5' terminal portion of the rat pro-alpha 1 (II) chain gene. These clones span about 20 kilobases (kb) of contiguous DNA containing 15 kb of the gene and 5 kb of the 5' flanking sequence. Electron microscopic analysis of mRNA-DNA hybrids by R-looping shows that collectively these clones contain 16 exons which code for approximately one-third of the pro-alpha 1 (II) chain. The sizes of the exons are small, except for the first exon which is relatively large. The nucleotide sequence of the first exon and the 1000 base pairs (bp) preceding it was determined. The first exon contains a 150-bp untranslated segment and an 85-bp sequence coding for the signal peptide and a part of the NH2-terminal propeptide of type II collagen. The segment preceding the transcription initiation site contains the "TATA" box and several G + C-rich stretches, whereas the "CAT" box is not evident between -70 and -120. The hexanucleotide sequence 5'-GGGCGG-3' is found in three different places between -200 and the TATA box. The inverted complement sequence of this hexanucleotide, 5'-CCGCCC-3', is located around both -220 and -450. The hexanucleotide and its inverted sequence have been found previously in the promoter region of the tk gene of herpes virus. These sequences are known to function in a mutually dependent manner as transcription signals for the tk gene; thus, they may play a role in determining the level of transcription of this cartilage gene. The hexanucleotide, 5'-CCGCCC-3', is also found in the 21-base pair repeats of the SV40 promoter and the promoter region of hydroxymethylglutaryl-CoA reductase gene. The sequence 5'-GTGGTTAGA-3' located around -280 is identical to the "core" sequence that has been reported as enhancer element in both viral and cellular genes. These unusual structures may be related to the tissue-specific expression of this gene.

Animals↗

PubMed1985

Rat hepatic cytosolic phosphoenolpyruvate carboxykinase (GTP). Structures of the protein, messenger RNA, and gene.

The primary structure of the messenger RNA coding for cytosolic phosphoenolpyruvate carboxykinase was determined by sequencing cDNA and genomic DNA and by primer extension of the mRNA. The molecule is 2624 nucleotides in length; this includes 143 nontranslated nucleotides at the 5' end and 615 nontranslated nucleotides at the 3' end. The 3' nontranslated sequence contains a 102-base pair region of alternating purine-pyrimidine nucleotides (the majority of which are UpG dinucleotides), several direct repeats and palindromic sequences, and 8 CpG dinucleotides. The corresponding segment of the phosphoenolpyruvate carboxykinase gene thus has characteristics which favor the formation of Z-DNA. The amino acid sequence of phosphoenolpyruvate carboxykinase was deduced from the mRNA sequence and confirmed by fast atom bombardment mass spectrometric analysis of peptides generated with trypsin and Staphylococcus aureus V8 protease. The protein consists of 621 amino acids and has a molecular weight of 69,289. Charon 4A lambda bacteriophage clones containing genomic DNA coding for phosphoenolpyruvate carboxykinase were isolated from a library of partial HaeIII digests of rat liver DNA. Two clones, lambda PC112 and lambda PC103, contained the entire coding region in 15-kilobase inserts and were used to subclone the gene into pBR322 as EcoRI, BamHI, or SstI-KpnI fragments. Using these subclones, the structure of the phosphoenolpyruvate carboxykinase gene was determined by S1 nuclease mapping, R-loop analysis, and DNA sequencing. The gene is composed of 10 exons and 9 introns with a total length of 6.0 kilobases. The transcription initiation site of the gene was determined by a combination of in vitro transcription in a HeLa cell lysate system, primer extension of mRNAPEPCK, and S1 nuclease mapping. In vitro transcription of purified DNA templates revealed three RNA polymerase II-dependent start sites. Two sites were separated by 600 base pairs on the coding strand and the third site was on the noncoding strand. The products of S1 nuclease mapping and primer extension from a BglII site were compared in order to determine which of the coding strand initiation sites was expressed in vivo. In both cases a 69-base pair fragment was generated and the 5' end of this corresponded to a thymidine residue identified in a sequence ladder of the genomic DNA coding strand. We conclude that mRNAPEPCK synthesis initiates with an adenine residue 69 base pairs 5' of the BglII site; this corresponds to the 3' most transcription initiation site determined in vitro.

Amino Acid Sequence↗

PubMed1986

Alpha 1 type IV collagen gene evolved differently from fibrillar collagen genes.

Type IV collagen is a major structural component in basement membranes. It is considerably different from the fibrillar collagens, types I-III. For example, unlike fibrillar collagens, the triple helical domain of type IV collagen is frequently interrupted by nonhelical regions. In this report, we demonstrate several overlapping genomic clones which cover most of the mouse alpha 1(IV) chain. Electron microscopic analysis of R-loops revealed that there were at least 28 exons within 35 kilobases of the gene segment. The sizes of six exons were determined by DNA sequence analysis to be 81, 178, 134, 73, 129, and 213 base pairs. These sizes do not appear to be related to the 54-base pair coding unit which is characteristic of fibrillar collagen exons, suggesting that the alpha 1 type IV collagen gene evolved differently from the fibrillar collagen genes.

Actin Cytoskeleton↗

PubMed1986

Characterization of the DNA of the hamster papovavirus: IV. Transcription mapping of calf-thymus DNA polymerase II.

Nascent RNA, synthesized by calf thymus RNA polymerase II on restriction endonuclease BamHI linearized hamster papovavirus (HaPV) DNA, was rehybridized to the template strand under conditions allowing transcription R-loop formation. Hybrids, visualized by electron microscopy, were plotted and mapped according to the physical map of HaPV. Two predominant regions of transcription could be localized at 0.10--0.40 and 0.50--0.82 m.u., respectively. For the start sites of transcription at map positions 0.67 and 0.75, respectively, on the HaPV genome a transcription in opposite direction were estimated. This genome region harbours the putative origin of replication of HaPV DNA. These results suggest a distinct relatedness of HaPV to the polyomavirus group.

Animals↗

PubMed1987

Structure of the Pseudomonas putida alkBAC operon. Identification of transcription and translation products.

The structural genes of the Pseudomonas oleovorans alk (alkane utilization) system, which are localized on the alkBAC operon, were cloned as a 16.9-kilobase pair EcoRI fragment. We have measured the length and determined the position of the alkBAC operon on this fragment by electron microscopy of R-loops. Furthermore, the 7.3-kilobase pair long alkBAC operon was analyzed for translation products in Escherichia coli minicells. Using a spectrum of overlapping subclones, six different proteins were identified. Starting from the alkBAC promotor, these polypeptides had molecular masses of 41, 15, 49, 58, 59, and 20 kDa, respectively. The 41-kDa protein was identified as alkane hydroxylase by reaction with a specific antibody. The 15- and 49-kDa peptides are soluble components of the alkane hydroxylase complex. The 58-kDa protein is most likely involved in alkanol dehydrogenase activity.

Alkanes↗

PubMed1988

Electron microscopy of nucleic acid.

Electron microscopy (EM) of nucleic acids was first described by Kleinschmidt and Zahn 1959 (1). By complexing duplex DNA with basic proteins and spreading the complexed DNA onto the surface of an aquous phase it was possible to obtain well stretched DNA molecules that could be examined by EM. Addition of formamide to DNA spreading solution as well as to hypophase prevented single-stranded DNA and RNA from forming secondary structures thus providing conditions for analysis of these molecules. EM studies have provided information about molecular weight, molecular configuration and homogeneity/heterogeneity of the DNA preparations examined. Furthermore, intramolecular heterogeneity in base sequence could be analyzed by partial denaturation of duplex DNA. By partial denaturation mapping information of the orientation of DNA molecules was obtained. Sequence homology between two DNA molecules were studied by heteroduplex analyses where the two DNAs to be studied were denatured to full strand separation followed by reassociation. This method has been widely used to analyze DNA mutations, and to discover sequence homologies within a DNA molecule or between two DNA molecules. Heteroduplex analyses between DNA and RNA (R-loop formation) provided information about distribution of ribosomal RNA genes and gave the first information of eukaryotic post transcriptional processing of mRNA. Also the various modes for DNA replication and transcription were studied by EM. It is possible to study duplex DNA not complexed by basic proteins by EM. The DNA is then adsorbed to an activated carbon film and rotary shadowed with finely grained metals to enhance the contrast. Using this method it was possible to study binding of proteins to DNA. This method was also used in the study of chromatin structure where it provided information of the size and distribution of nucleosomes as well as packaging of DNA in chromatin fibres. The complicated nature of DNA strand exchange and repair reactions was studied by in-vitro systems where EM analysis was used in the description of the presynoptic filament and structure of the joined molecules. Furthermore, EM provided the first direct evidence for the existence of the paranemic joint, where the DNA strands are joined without Watson-Crick base-pairing.

Animals↗

PubMed1979

Isolation and characterization of recombinant clones containing the chicken adult beta-globin gene.

We have isolated and characterized two independent clones containing the chicken adult beta-globin gene. Each clone contains a 6.2-kilobase-pair Eco RI restriction fragment of chicken erythrocyte DNA inserted into the vector, lambda gtWES . lambda B. The orientation of the inserted fragment is opposite in the two clones. Characterization of the clones by electron microscopic R-loop studies, by restriction enzyme mapping, and by filter hybridization shows that the adult beta-globin gene is interrupted by at least one small and one large intervening sequence. In addition to the complete adult beta-globin gene, at least part of a second beta-globin-like gene was identified about 2.7 kilobase pairs from the 3'-end of the adult gene. The two independent clones, while very similar, do differ at two Msp I restriction endonuclease sites in regions flanking the adult beta-globin gene.

Animals↗

PubMed1985

The myosin alkali light chains of mouse ventricular and slow skeletal muscle are indistinguishable and are encoded by the same gene.

We have isolated a cDNA recombinant plasmid (pA29) identified as encoding part of the ventricular muscle myosin light chain MLC1v. This cDNA contains a 300-base pair fragment which under conditions of moderate stringency shows specific hybridization to MLC1v mRNA with no detectable cross-hybridization with the mRNAs encoding the fast skeletal muscle isoforms MLC1F and MLC3F, or the atrial muscle isoform MLC1A. Under these conditions hybridization is seen with an abundant mRNA present in slow skeletal muscle (soleus) which is indistinguishable from ventricular MLC1V mRNA on the basis of size and of thermal stability of hybrids formed with plasmid pA29. The mouse MLC1V and MLC1S proteins are found to co-migrate on two-dimensional gels. We therefore conclude that these isoforms are the same and are encoded by the same mRNA. Analysis of mouse DNA has identified a single region of the genome which hybridizes to this same fragment of pA29. This region has been isolated in a recombinant phage and has been shown to contain a single gene showing homology with MLC1V mRNA by R-loop analysis. We therefore conclude that MLC1V and MLC1S are encoded by a single gene. The pattern of segregation of a restriction fragment length polymorphism identified for this gene between Mus musculus and Mus spretus has been followed in an F1 backcross between these two mouse species. The results show the MLC1V/MLC1S gene to be closely linked to a marker at the distal end of mouse chromosome 9.

Animals↗